IL-1 eta DNA and polypeptides

ABSTRACT

The invention is directed to novel, purified and isolated IL-1 eta polypeptides and fragments thereof, the polynucleotides encoding such polypeptides, processes for production of recombinant forms of such polypeptides, antibodies generated against these polypeptides, peptides derived from these polypeptides, and uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of Ser. No. 09/976,472, filed on Oct.11, 2001 now U.S. Pat. No. 6,753,166; which is a continuation-in-part ofPCT application Ser. No. PCT/US00/14435 filed May 25, 2000; which claimsbenefit of provisional applications Ser. No. 60/162,331 filed Oct. 29,1999; and Ser. No. 60/135,758 filed May 25, 1999.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention is directed to novel, purified and isolated IL-1 etapolypeptides and fragments thereof, the polynucleotides encoding suchpolypeptides, processes for production of recombinant forms of suchpolypeptides, antibodies generated against these polypeptides,fragmented peptides derived from these polypeptides, and uses thereof.

2. Description of Related Art

Interleukin-1 (IL-1) is a member of a large group of cytokines whoseprimary function is to mediate immune and inflammatory responses. Thereare seven known IL-1 family members which include IL-1 alpha (IL-1α),IL-1 beta (IL-1β), IL-1 receptor antagonist (IL-1ra), IL-1 delta(IL-1δ), IL-1 epsilon (IL-1ε), IL-1 zeta (IL-1ξ) and IL-18 (previouslyknown as IGIF and sometimes IL-1 gamma). IL-1 that is secreted bymacrophages is actually a mixture of mostly IL-1β and some IL-1α (Abbaset al., 1994). IL-1α and IL-1β, which are first produced as 33 kDprecursors that lack a signal sequence, are further processed byproteolytic cleavage to produce secreted active forms, each about 17 kD.Additionally, the 33 kD precursor of IL-1α is also active. Both forms ofIL-1 are the products of two different genes located on chromosome 2.Although the two forms are less than 30 percent homologous to eachother, they both bind to the same receptors and have similar activities.

IL-1ra, a biologically inactive form of IL-1, is structurally homologousto IL-1 and binds to the same receptors. Additionally, IL-1ra isproduced with a signal sequence which allows for efficient secretioninto the extracellular region where it competitively competes with IL-1(Abbas et al., 1994).

The IL-1 family of ligands binds to a family of two IL-1 receptors,which are members of the Ig superfamily. IL-1 receptors include the 80kDa type I receptor (IL-1RI) and a 68 kDa type II receptor (IL-1RII).IL-1 ligands can also bind to a soluble proteolytic fragment of IL-1RII(sIL-1RII) (Colotta et al., 1993).

The major source of IL-1 is the activated macrophage or mononuclearphagocyte. Other cells that produce IL-1 include epithelial andendothelial cells (Abbas et al., 1994). IL-1 secretion from macrophagesoccurs after the macrophage encounters and ingests gram-negativebacteria. Such bacteria contain lipopolysaccharide (LPS) molecules, alsoknown as endotoxin, in the bacterial cell wall. LPS molecules are theactive components that stimulate macrophages to produce tumor necrosisfactor (TNF) and IL-1. In this case, IL-1 is produced in response to LPSand TNF production. At low concentrations, LPS stimulates macrophagesand activates B-cells and other host responses needed to eliminate thebacterial infection; however, at high concentrations, LPS can causesevere tissue damage, shock, and even death.

The biological functions of IL-1 include activating vascular endothelialcells and lymphocytes, local tissue destruction, and fever (Janeway etal., 1996). At low levels, IL-1 stimulates macrophages and vascularendothelial cells to produce IL-6, upregulates molecules on the surfaceof vascular endothelial cells to increase leukocyte adhesion, andindirectly activates inflammatory leukocytes by stimulating mononuclearphagocytes and other cells to produce certain chemokines that activateinflammatory leukocytes. Additionally, IL-1 is involved in otherinflammatory responses such as induction of prostaglandins, nitric oxidesynthetase, and metalloproteinases. These IL-1 functions are crucialduring low level microbial infections. However, if the microbialinfection escalates, IL-1 acts systemically by inducing fever,stimulating mononuclear phagocytes to produce IL-1 and IL-6, increasingthe production of serum proteins from hepatocytes, and activating thecoagulation system. Additionally, IL-1 does not cause hemorrhagicnecrosis of tumors, suppress bone marrow stem cell division, and IL-1 islethal to humans at high concentrations.

Given the important function of IL-1, there is a need in the art foradditional members of the IL-1 ligand and IL-1 receptor families. Inaddition, in view of the continuing interest in protein research and theimmune system, the discovery, identification, and roles of new proteins(such as the human IL-1 eta of the invention) and their inhibitors, areat the forefront of modern molecular biology and biochemistry. Despitethe growing body of knowledge, there is still a need in the art for theidentity and function of proteins involved in cellular and immuneresponses.

SUMMARY OF THE INVENTION

The invention aids in fulfilling these various needs in the art byproviding isolated polynucleotides and polypeptides encoded by thepolynucleotides for the novel IL-1 family ligand termed “IL-1 eta.”Thus, in one aspect, the invention is directed to isolated novelpolynucleotide molecules of IL-1 eta comprising the nucleotide residues112–585 of SEQ ID NO:1 and to the isolated polynucleotide moleculesencoding the amino acid sequence of SEQ ID NO:2, as well aspolynucleotide molecules complementary to these sequences.

Both single-stranded and double-stranded RNA and DNA molecules areencompassed by the invention, as well as polynucleotide molecules thathybridize to a denatured, double-stranded DNA comprising all or aportion of SEQ ID NO:1 and/or a DNA that encodes the amino acidsequences set forth in SEQ ID NO:2. Also encompassed are isolatedpolynucleotide molecules that are derived by in vitro mutagenesis ofpolynucleotide molecules comprising the coding region of SEQ ID NO:1,that are degenerate from polynucleotide molecules comprising thesequence of SEQ ID NO:1, and that are allelic variants of DNA of theinvention. The invention also encompasses recombinant vectors thatdirect the expression of these polynucleotide molecules and host cellstransformed or transfected with these vectors.

In addition, the invention encompasses methods of using the DNA notedabove to identify DNA encoding proteins having activities associatedwith IL-1 family ligands and receptors.

In addition, these polynucleotides can be used to identify the humanchromosomes with which the polynucleotides are associated. Thus, sincethe IL-1 eta polynucleotides map to chromosome 2, DNA encoding IL-1 etapolypeptides may be used to identify human chromosome 2. Accordingly,these polynucleotides may also be used to map genes on human chromosome2; to identify genes associated with certain diseases, syndromes, orother human conditions associated with human chromosome 2; and to studycell signal transduction and the immune system.

The invention also encompasses the use of sense or antisenseoligonucleotides from the polynucleotides of SEQ ID NO:1 to inhibit theexpression of the respective polynucleotide encoded by the genes of theinvention.

The invention also encompasses isolated polypeptides and fragments ofIL-1 eta as encoded by these polynucleotide molecules, including solublepolypeptide portions of SEQ ID NO:2. The invention further encompassesmethods for the production of these polypeptides, including culturing ahost cell under conditions promoting expression and recovering thepolypeptide from the culture medium. Especially, the expression of thesepolypeptides in bacteria, yeast, plant, insect, and animal cells isencompassed by the invention.

In general, the polypeptides of the invention can be used to studycellular processes such as immune regulation, cell proliferation, celldeath, cell migration, cell-to-cell interaction, and inflammatoryresponses. In addition, these polypeptides can be used to identifyproteins associated with IL-1 eta ligands.

In addition, the invention includes assays utilizing these polypeptidesto screen for potential inhibitors of activity associated withpolypeptide counter-structure molecules, and methods of using thesepolypeptides as therapeutic agents for the treatment of diseasesmediated by polypeptide counter-structure molecules. Further, methods ofusing these polypeptides in the design of inhibitors (e.g., engineeredreceptors that act as inhibitors) thereof are also an aspect of theinvention.

Further encompassed by this invention is the use of the IL-1 etapolynucleotide sequences, predicted amino acid sequences of thepolypeptide or fragments thereof, or a combination of the predictedamino acid sequences of the polypeptide and fragments thereof for use insearching an electronic database to aid in the identification of samplepolynucleotides and/or proteins.

Isolated polyclonal or monoclonal antibodies that bind to thesepolypeptides are also encompassed by the invention, in addition the useof these antibodies to aid in purifying the polypeptides of theinvention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 presents the nucleotide sequence of IL-1 eta (SEQ ID NO:1).

FIG. 2 presents the amino acid sequence of IL-1 eta (SEQ ID NO:2).

FIG. 3 is a table summarizing expression data of IL-1 eta. “-” indicatesthat the mRNA was looked for but not found; a blank space indicates thatthe analysis was not done for that particular gene/RNA combination.

DETAILED DESCRIPTION OF THE INVENTION

The polynucleotide molecules encompassed in the invention include thefollowing nucleotide sequence:

Name: IL-1 eta 1 GGCACGAGGT TCCTCCCCAC TCTGTCTTTC TCACCTCTCC TTCACTTTTC(SEQ ID NO:1) 51 CTAGCCTCCT CACCACCATC TGATCTATCT TGTTCTCTTC ACAAAAGGCT101 CTGAAGACAT CATGAACCCA CAACGGGAGG CAGCACCCAA ATCCTATGCT 151ATTCGTGATT CTCGACAGAT GGTGTGGGTC CTGAGTGGAA ATTCTTTAAT 201 AGCAGCTCCTCTTAGCCGCA GCATTAAGCC TGTCACTCTT CATTTAATAG 251 CCTGTAGAGA CACAGAATTCAGTGACAAGG AAAAGGGTAA TATGGTTTAC 301 CTGGGAATCA AGGGAAAAGA TCTCTGTCTCTTCTGTGCAG AAATTCAGGG 351 CAAGCCTACT TTGCAGCTTA AGGAAAAAAA TATCATGGACCTGTATGTGG 401 AGAAGAAAGC ACAGAAGCCC TTTCTCTTTT TCCACAATAA AGAAGGCTCC451 ACTTCTGTCT TTCAGTCAGT CTCTTACCCT GGCTGGTTCA TAGCCACCTC 501CACCACATCA GGACAGCCCA TCTTTCTCAC CAAGGAGAGA GGCATAACTA 551 ATAACACTAACTTCTACTTA GATTCTGTGG AATAA

The amino acid sequence of the polypeptides encoded by the nucleotidesequence of the invention include:

Name: IL-1 eta (polypeptide) 1 MNPQREAAPK SYAIRDSRQM VWVLSGNSLIAAPLSRSIKP VTLHLIACRD (SEQ ID NO:2) 51 TEFSDKEKGN MVYLGIKGKD LCLFCAEIQGKPTLQLKEKN IMDLYVEKKA 101 QKPFLFFHNK EGSTSVFQSV SYPGWFIATS TTSGQPIFLTKERGITNNTN 151 FYLDSVE*

The discovery of the IL-1 eta polynucleotides of the invention enablesthe construction of expression vectors comprising polynucleotidesequences encoding the respective polypeptides and host cellstransfected or transformed with the expression vectors. The inventionalso enables the isolation and purification of biologically active IL-1eta polypeptides and fragments thereof. In yet another embodiment, thepolynucleotides or oligonucleotides thereof can be used as probes toidentify DNA encoding proteins having activities associated with IL-1family members. In addition, the polynucleotides or oligonucleotidesthereof of the present invention may be used to identify humanchromosome 2. Similarly, the polynucleotides or oligonucleotides thereofof the present invention may be used to map genes on human chromosome 2,and to identify genes associated with certain diseases, syndromes orother human conditions associated with human chromosome 2. Among suchdiseases, syndromes or conditions are glaucoma, ectodermal dysplasia,insulin-dependent diabetes mellitus, wrinkly skin syndrome, T-cellleukemia/lymphoma, and tibial muscular dystrophy. Finally,single-stranded sense or antisense oligonucleotides from thesepolynucleotides can be used to inhibit expression of polynucleotidesencoded by the IL-1 eta.

Further, and in accordance with the present invention, IL-1 etapolypeptides and/or soluble fragments thereof, can be used to activateand/or inhibit the activation of vascular endothelial cells andlymphocytes, induce and/or inhibit the induction of local tissuedestruction and fever (Janeway et al., 1996), inhibit and/or stimulatemacrophages and vascular endothelial cells to produce IL-6, induceand/or inhibit the induction of prostaglandins, nitric oxide synthetase,and metalloproteinases, and upregulate and/or inhibit the upregulationof molecules on the surface of vascular endothelial cells. In additionthese polypeptides and fragmented peptides can also be used to induceand/or inhibit the induction of inflammatory mediators such astranscription factors NF-κB and AP-1, MAP kinases JNK and p38, COX-2,iNOS, and all of the activities stimulated by these molecules. Thepolypeptides of this invention, and fragments thereof, can be used togenerate antibodies, and the invention includes the use of suchantibodies to purify IL-1 eta polypeptides.

Polynucleotide Molecules

In one embodiment, the present invention involves certainpolynucleotides that are free from contaminating endogenous material. Apolynucleotide refers to a DNA molecule in the form of a separatefragment or as a component of a larger polynucleotide construct. Thepolynucleotide molecule has been derived from DNA or RNA isolated atleast once in a quantity that allows identification of its componentnucleotide sequence by standard biochemical methods (such as thoseoutlined in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nded., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989)).Such sequences are preferably provided and/or constructed in the form ofan open reading frame uninterrupted by internal non-translatedsequences, or introns, that are typically present in eukaryotic genes.Sequences of non-translated DNA can be present 5′ or 3′ from an openreading frame, where the same do not interfere with manipulation orexpression of the coding region. In one embodiment of the instantinvention, the open reading frame runs from nucleotide 112 to the TAAstop codon.

Polynucleotide molecules of the invention include DNA in bothsingle-stranded and double-stranded form, as well as the RNA complementthereof. DNA includes, for example, cDNA, genomic DNA, chemicallysynthesized DNA, DNA amplified by PCR, and combinations thereof. GenomicDNA may be isolated by conventional techniques, e.g., using the cDNA ofSEQ ID NO:1, or a suitable fragment thereof, as a probe.

The DNA molecules of the invention include full length genes as well aspolynucleotides and fragments thereof. The full length gene may includethe N-terminal signal peptide. Other embodiments include DNA encoding asoluble form, e.g., encoding the extracellular domain of the protein,either with or without the signal peptide.

The polynucleotides of the invention are preferentially derived fromhuman sources, but the invention includes those derived from non-humanspecies, as well.

The particularly preferred polynucleotide of the invention has thepolynucleotide sequence shown in the encoding region of SEQ ID NO:1(beginning at nucleotide 112), for IL-1 eta. cDNA clones having thenucleotide sequence of SEQ ID NO:1 were isolated as described inExample 1. The polypeptide encoded by the IL-1 eta DNA of SEQ ID NO:1 isshown in SEQ ID NO:2.

The polypeptide of SEQ ID NO:2 shares homology with other IL-1 familymembers.

Due to the known degeneracy of the genetic code, wherein more than onecodon can encode the same amino acid, a DNA can vary from that shown inSEQ ID NO:1, and still encode a polypeptide having the amino acidsequence of SEQ ID NO:2. Such variant DNA sequences can result fromsilent mutations (e.g., occurring during PCR amplification), or can bethe product of deliberate mutagenesis of a native sequence.

The invention thus provides isolated DNA sequences encoding polypeptidesof the invention, selected from: (a) DNA comprising the nucleotideresides 112–585 of SEQ ID NO:1; (b) DNA encoding the polypeptides of SEQID NO:2; (c) DNA that is the complement of DNA that is capable ofhybridization to a DNA of (a) or (b) under conditions of moderatestringency and which encodes polypeptides of the invention; (d) DNA thatis the complement of DNA capable of hybridization to a DNA of (a) or (b)under conditions of high stringency and which encodes polypeptides ofthe invention, and (e) DNA which is degenerate, as a result of thegenetic code, to a DNA defined in (a), (b), (c), or (d) and which encodepolypeptides of the invention. Of course, polypeptides encoded by suchDNA sequences are encompassed by the invention.

As used herein, conditions of moderate stringency can be readilydetermined by those having ordinary skill in the art based on, forexample, the length of the DNA. The basic conditions are set forth bySambrook et al. Molecular Cloning: A Laboratory Manual, 2 ed. Vol. 1,pp. 1.101–104, Cold Spring Harbor Laboratory Press, (1989), and includeuse of a prewashing solution for the nitrocellulose filters 5×SSC, 0.5%SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of about 50%formamide, 6×SSC at about 42° C. (or other similar hybridizationsolution, such as Stark's solution, in about 50% formamide at about 42°C.), and washing conditions of about 60° C., 0.5×SSC, 0.1% SDS.Conditions of high stringency can also be readily determined by theskilled artisan based on, for example, the length of the DNA. Generally,such conditions are defined as hybridization conditions as above, andwith washing at approximately 68° C., 0.2×SSC, 0.1% SDS. The skilledartisan will recognize that the temperature and wash solution saltconcentration can be adjusted as necessary according to factors such asthe length of the probe.

Also included as an embodiment of the invention is DNA encodingpolypeptide fragments and polypeptides comprising inactivatedN-glycosylation site(s), inactivated protease processing site(s), orconservative amino acid substitution(s), as described below.

In another embodiment, the polynucleotide molecules of the inventionalso comprise nucleotide sequences that are at least 80% identical to anative sequence. Also contemplated are embodiments in which apolynucleotide molecule comprises a sequence that is at least 90%identical, at least 95% identical, at least 98% identical, at least 99%identical, or at least 99.9% identical to a native sequence.

The percent identity may be determined by visual inspection andmathematical calculation. Alternatively, the percent identity of twopolynucleotide sequences can be determined by comparing sequenceinformation using the GAP computer program, version 6.0 described byDevereux et al. (Nucl. Acids Res. 12:387, 1984) and available from theUniversity of Wisconsin Genetics Computer Group (UWGCG). The preferreddefault parameters for the GAP program include: (1) a unary comparisonmatrix (containing a value of 1 for identities and 0 for non-identities)for nucleotides, and the weighted comparison matrix of Gribskov andBurgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz andDayhoff, eds., Atlas of Protein Sequence and Structure, NationalBiomedical Research Foundation, pp. 353–358, 1979; (2) a penalty of 3.0for each gap and an additional 0.10 penalty for each symbol in each gap;and (3) no penalty for end gaps. Other programs used by one skilled inthe art of sequence comparison may also be used.

The invention provides isolated polynucleotides useful in the productionof polypeptides. Such polypeptides may be prepared by any of a number ofconventional techniques. A DNA sequence encoding a polypeptide of theinvention, or desired fragment thereof may be subcloned into anexpression vector for production of the polypeptide or fragment. The DNAsequence advantageously is fused to a sequence encoding a suitableleader or signal peptide. Alternatively, the desired fragment may bechemically synthesized using known techniques. DNA fragments also may beproduced by restriction endonuclease digestion of a full length clonedDNA sequence, and isolated by electrophoresis on agarose gels. Ifnecessary, oligonucleotides that reconstruct the 5′ or 3′ terminus to adesired point may be ligated to a DNA fragment generated by restrictionenzyme digestion. Such oligonucleotides may additionally contain arestriction endonuclease cleavage site upstream of the desired codingsequence, and position an initiation codon (ATG) at the N-terminus ofthe coding sequence.

The well-known polymerase chain reaction (PCR) procedure also may beemployed to isolate and amplify a DNA sequence encoding a desiredprotein fragment. Oligonucleotides that define the desired termini ofthe DNA fragment are employed as 5′ and 3′ primers. The oligonucleotidesmay additionally contain recognition sites for restrictionendonucleases, to facilitate insertion of the amplified DNA fragmentinto an expression vector. PCR techniques are described in Saiki et al.,Science 239:487 (1988); Recombinant DNA Methodology, Wu et al., eds.,Academic Press, Inc., San Diego (1989), pp. 189–196; and PCR Protocols:A Guide to Methods and Applications, Innis et al., eds., Academic Press,Inc. (1990).

Polypeptides and Fragments Thereof

The invention encompasses polypeptides and fragments thereof in variousforms, including those that are naturally occurring or produced throughvarious techniques such as procedures involving recombinant DNAtechnology. Such forms include, but are not limited to, derivatives,variants, and oligomers, as well as fusion proteins or fragmentsthereof.

The polypeptides of the invention include full length proteins encodedby the polynucleotide sequences set forth above. Particularly preferredpolypeptides of IL-1 eta comprise the amino acid sequence of SEQ IDNO:2.

The polypeptides of the invention may be secreted and, thus, soluble.Soluble polypeptides are capable of being secreted from the cells inwhich they are expressed. In general, soluble polypeptides may beidentified (and distinguished from non-soluble membrane-boundcounterparts) by separating intact cells which express the desiredpolypeptide from the culture medium, e.g., by centrifugation, andassaying the medium (supernatant) for the presence of the desiredpolypeptide. The presence of polypeptide in the medium indicates thatthe polypeptide was secreted from the cells and thus is a soluble formof the protein.

In one embodiment, the soluble polypeptides and fragments thereofcomprise all or part of the extracellular domain, but lack thetransmembrane region that would cause retention of the polypeptide on acell membrane. A soluble polypeptide may include the cytoplasmic domain,or a portion thereof, as long as the polypeptide is secreted from thecell in which it is produced.

In general, the use of soluble forms is advantageous for certainapplications. Purification of the polypeptides from recombinant hostcells is facilitated, since the soluble polypeptides are secreted fromthe cells. Further, soluble polypeptides are generally more suitable forintravenous administration.

The invention also provides polypeptides and fragments of theextracellular domain that retain a desired biological activity.Particular embodiments are directed to polypeptide fragments of SEQ IDNO:2 that retain the ability to bind the native cognates, substrates, orcounter-structure (“binding partner”). Such a fragment may be a solublepolypeptide, as described above. In another embodiment, the polypeptidesand fragments advantageously include regions that are conserved in theIL-1 ligand and IL-1 receptor family as described above.

Also provided herein are polypeptide fragments comprising at least 20,or at least 30, contiguous amino acids of the sequences of SEQ ID NO:2.Polypeptide fragments also may be employed as immunogens, in generatingantibodies.

Naturally occurring variants as well as derived variants of thepolypeptides and fragments are provided herein. Variants may exhibitamino acid sequences that are at least 80% identical. Also contemplatedare embodiments in which a polypeptide or fragment comprises an aminoacid sequence that is at least 90% identical, at least 95% identical, atleast 98% identical, at least 99% identical, or at least 99.9% identicalto the preferred polypeptide or fragment thereof. Percent identity maybe determined by visual inspection and mathematical calculation.Alternatively, the percent identity of two protein sequences can bedetermined by comparing sequence information using the GAP computerprogram, based on the algorithm of Needleman and Wunsch (J. Mol. Bio.48:443, 1970) and available from the University of Wisconsin GeneticsComputer Group (UWGCG). The preferred default parameters for the GAPprogram include: (1) a scoring matrix, blosum62, as described byHenikoff and Henikoff (Proc. Natl. Acad. Sci. USA 89:10915, 1992); (2) agap weight of 12; (3) a gap length weight of 4; and (4) no penalty forend gaps. Other programs used by one skilled in the art of sequencecomparison may also be used.

The variants of the invention include, for example, those that resultfrom alternate mRNA splicing events or from proteolytic cleavage.Alternate splicing of mRNA may, for example, yield a truncated butbiologically active protein, such as a naturally occurring soluble formof the protein. Variations attributable to proteolysis include, forexample, differences in the N- or C-termini upon expression in differenttypes of host cells, due to proteolytic removal of one or more terminalamino acids from the protein (generally from 1–5 terminal amino acids).Proteins in which differences in amino acid sequence are attributable togenetic polymorphism (allelic variation among individuals producing theprotein) are also contemplated herein.

Additional variants within the scope of the invention includepolypeptides that may be modified to create derivatives thereof byforming covalent or aggregative conjugates with other chemical moieties,such as glycosyl groups, lipids, phosphate, acetyl groups and the like.Covalent derivatives may be prepared by linking the chemical moieties tofunctional groups on amino acid side chains or at the N-terminus orC-terminus of a polypeptide. Conjugates comprising diagnostic(detectable) or therapeutic agents attached thereto are contemplatedherein, as discussed in more detail below.

Other derivatives include covalent or aggregative conjugates of thepolypeptides with other proteins or polypeptides, such as by synthesisin recombinant culture as N-terminal or C-terminal fusions. Examples offusion proteins are discussed below in connection with oligomers.Further, fusion proteins can comprise peptides added to facilitatepurification and identification. Such peptides include, for example,poly-His or the antigenic identification peptides described in U.S. Pat.No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988. One suchpeptide is the FLAG® peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, which ishighly antigenic and provides an epitope reversibly bound by a specificmonoclonal antibody, enabling rapid assay and facile purification ofexpressed recombinant protein. A murine hybridoma designated 4E11produces a monoclonal antibody that binds the FLAG® peptide in thepresence of certain divalent metal cations, as described in U.S. Pat.No. 5,011,912, hereby incorporated by reference. The 4E11 hybridoma cellline has been deposited with the American Type Culture Collection underaccession no. HB 9259. Monoclonal antibodies that bind the FLAG® peptideare available from Eastman Kodak Co., Scientific Imaging SystemsDivision, New Haven, Conn.

Among the variant polypeptides provided herein are variants of nativepolypeptides that retain the native biological activity or thesubstantial equivalent thereof. One example is a variant that binds withessentially the same binding affinity as does the native form. Bindingaffinity can be measured by conventional procedures, e.g., as describedin U.S. Pat. No. 5,512,457 and as set forth below.

Variants include polypeptides that are substantially homologous to thenative form, but which have an amino acid sequence different from thatof the native form because of one or more deletions, insertions orsubstitutions. Particular embodiments include, but are not limited to,polypeptides that comprise from one to ten deletions, insertions orsubstitutions of amino acid residues, when compared to a nativesequence.

A given amino acid may be replaced, for example, by a residue havingsimilar physiochemical characteristics. Examples of such conservativesubstitutions include substitution of one aliphatic residue for another,such as Ile, Val, Leu, or Ala for one another; substitutions of onepolar residue for another, such as between Lys and Arg, Glu and Asp, orGln and Asn; or substitutions of one aromatic residue for another, suchas Phe, Trp, or Tyr for one another. Other conservative substitutions,e.g., involving substitutions of entire regions having similarhydrophobicity characteristics, are well known.

Similarly, the DNAs of the invention include variants that differ from anative DNA sequence because of one or more deletions, insertions orsubstitutions, but that encode a biologically active polypeptide.

The invention further includes polypeptides of the invention with orwithout associated native-pattern glycosylation. Polypeptides expressedin yeast or mammalian expression systems (e.g., COS-1 or COS-7 cells)can be similar to or significantly different from a native polypeptidein molecular weight and glycosylation pattern, depending upon the choiceof expression system. Expression of polypeptides of the invention inbacterial expression systems, such as E. coli, provides non-glycosylatedmolecules. Further, a given preparation may include multipledifferentially glycosylated species of the protein. Glycosyl groups canbe removed through conventional methods, in particular those utilizingglycopeptidase. In general, glycosylated polypeptides of the inventioncan be incubated with a molar excess of glycopeptidase (BoehringerMannheim).

Correspondingly, similar DNA constructs that encode various additions orsubstitutions of amino acid residues or sequences, or deletions ofterminal or internal residues or sequences are encompassed by theinvention. For example, N-glycosylation sites in the polypeptideextracellular domain can be modified to preclude glycosylation, allowingexpression of a reduced carbohydrate analog in mammalian and yeastexpression systems. N-glycosylation sites in eukaryotic polypeptides arecharacterized by an amino acid triplet Asn-X-Y, wherein X is any aminoacid except Pro and Y is Ser or Thr. Appropriate substitutions,additions, or deletions to the nucleotide sequence encoding thesetriplets will result in prevention of attachment of carbohydrateresidues at the Asn side chain. Alteration of a single nucleotide,chosen so that Asn is replaced by a different amino acid, for example,is sufficient to inactivate an N-glycosylation site. Alternatively, theSer or Thr can by replaced with another amino acid, such as Ala. Knownprocedures for inactivating N-glycosylation sites in proteins includethose described in U.S. Pat. No. 5,071,972 and EP 276,846, herebyincorporated by reference.

In another example of variants, sequences encoding Cys residues that arenot essential for biological activity can be altered to cause the Cysresidues to be deleted or replaced with other amino acids, preventingformation of incorrect intramolecular disulfide bridges upon folding orrenaturation.

Other variants are prepared by modification of adjacent dibasic aminoacid residues, to enhance expression in yeast systems in which KEX2protease activity is present. EP 212,914 discloses the use ofsite-specific mutagenesis to inactivate KEX2 protease processing sitesin a protein. KEX2 protease processing sites are inactivated bydeleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, andLys-Arg pairs to eliminate the occurrence of these adjacent basicresidues. Lys-Lys pairings are considerably less susceptible to KEX2cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents aconservative and preferred approach to inactivating KEX2 sites.

Oligomers

Encompassed by the invention are oligomers or fusion proteins thatcontain IL-1 eta polypeptides. Such oligomers may be in the form ofcovalently linked or non-covalently-linked multimers, including dimers,trimers, or higher oligomers. As noted above, preferred polypeptides aresoluble and thus these oligomers may comprise soluble polypeptides. Inone aspect of the invention, the oligomers maintain the binding abilityof the polypeptide components and provide therefor, bivalent, trivalent,etc., binding sites.

One embodiment of the invention is directed to oligomers comprisingmultiple polypeptides joined via covalent or non-covalent interactionsbetween peptide moieties fused to the polypeptides. Such peptides may bepeptide linkers (spacers), or peptides that have the property ofpromoting oligomerization. Leucine zippers and certain polypeptidesderived from antibodies are among the peptides that can promoteoligomerization of the polypeptides attached thereto, as described inmore detail below.

Immunoglobulin-Based Oligomers

As one alternative, an oligomer is prepared using polypeptides derivedfrom immunoglobulins. Preparation of fusion proteins comprising certainheterologous polypeptides fused to various portions of antibody-derivedpolypeptides (including the Fc domain) has been described, e.g., byAshkenazi et al. (PNAS USA 88:10535, 1991); Byrn et al. (Nature 344:677,1990); and Hollenbaugh and Aruffo (“Construction of ImmunoglobulinFusion Proteins”, in Current Protocols in Immunology, Suppl. 4, pages10.19.1–10.19.11, 1992).

One embodiment of the present invention is directed to a dimercomprising two fusion proteins created by fusing a polypeptide of theinvention to an Fc polypeptide derived from an antibody. A gene fusionencoding the polypeptide/Fc fusion protein is inserted into anappropriate expression vector. Polypeptide/Fc fusion proteins areexpressed in host cells transformed with the recombinant expressionvector, and allowed to assemble much like antibody molecules, whereuponinterchain disulfide bonds form between the Fc moieties to yielddivalent molecules.

The term “Fc polypeptide” as used herein includes native and muteinforms of polypeptides made up of the Fc region of an antibody comprisingany or all of the CH domains of the Fc region. Truncated forms of suchpolypeptides containing the hinge region that promotes dimerization arealso included. Preferred polypeptides comprise an Fc polypeptide derivedfrom a human IgG1 antibody.

One suitable Fc polypeptide, described in PCT application WO 93/10151,hereby incorporated by reference, is a single chain polypeptideextending from the N-terminal hinge region to the native C-terminus ofthe Fc region of a human IgG1 antibody. Another useful Fc polypeptide isthe Fc mutein described in U.S. Pat. No. 5,457,035 and in Baum et al.,(EMBO J. 13:3992–4001, 1994) incorporated herein by reference. The aminoacid sequence of this mutein is identical to that of the native Fcsequence presented in WO 93/10151, except that amino acid 19 has beenchanged from Leu to Ala, amino acid 20 has been changed from Leu to Glu,and amino acid 22 has been changed from Gly to Ala. The mutein exhibitsreduced affinity for Fc receptors.

The above-described fusion proteins comprising Fc moieties (andoligomers formed therefrom) offer the advantage of facile purificationby affinity chromatography over Protein A or Protein G columns.

In other embodiments, the polypeptides of the invention may besubstituted for the variable portion of an antibody heavy or lightchain. If fusion proteins are made with both heavy and light chains ofan antibody, it is possible to form an oligomer with as many as fourpolypeptide extracellular region.

Alternatively, the oligomer is a fusion protein comprising multiplepolypeptides, with or without peptide linkers (spacer peptides). Amongthe suitable peptide linkers are those described in U.S. Pat. Nos.4,751,180 and 4,935,233, which are hereby incorporated by reference. ADNA sequence encoding a desired peptide linker may be inserted between,and in the same reading frame as, the DNA sequences of the invention,using any suitable conventional technique. For example, a chemicallysynthesized oligonucleotide encoding the linker may be ligated betweenthe sequences. In particular embodiments, a fusion protein comprisesfrom two to four soluble polypeptides of the invention, separated bypeptide linkers.

Another method for preparing the oligomers of the invention involves useof a leucine zipper. Leucine zipper domains are peptides that promoteoligomerization of the proteins in which they are found. Leucine zipperswere originally identified in several DNA-binding proteins (Landschulzet al., Science 240:1759, 1988), and have since been found in a varietyof different proteins. Among the known leucine zippers are naturallyoccurring peptides and derivatives thereof that dimerize or trimerize.

The zipper domain (also referred to herein as an oligomerizing, oroligomer-forming, domain) comprises a repetitive heptad repeat, oftenwith four or five leucine residues interspersed with other amino acids.Examples of zipper domains are those found in the yeast transcriptionfactor GCN4 and a heat-stable DNA-binding protein found in rat liver(C/EBP; Landschulz et al., Science 243:1681, 1989). Two nucleartransforming proteins, fos and jun, also exhibit zipper domains, as doesthe gene product of the murine proto-oncogene, c-myc (Landschulz et al.,Science 240:1759, 1988). The products of the nuclear oncogenes fos andjun comprise zipper domains that preferentially form heterodimers(O'Shea et al., Science 245:646, 1989, Turner and Tjian, Science243:1689, 1989). The zipper domain is necessary for biological activity(DNA binding) in these proteins.

The fusogenic proteins of several different viruses, includingparamyxovirus, coronavirus, measles virus and many retroviruses, alsopossess zipper domains (Buckland and Wild, Nature 338:547,1989; Britton,Nature 353:394, 1991; Delwart and Mosialos, AIDS Research and HumanRetroviruses 6:703, 1990). The zipper domains in these fusogenic viralproteins are near the transmembrane region of the proteins; it has beensuggested that the zipper domains could contribute to the oligomericstructure of the fusogenic proteins. Oligomerization of fusogenic viralproteins is involved in fusion pore formation (Spruce et al, Proc. Natl.Acad. Sci. U.S.A. 88:3523, 1991). Zipper domains have also been reportedrecently to play a role in oligomerization of heat-shock transcriptionfactors (Rabindran et al., Science 259:230, 1993).

Zipper domains fold as short, parallel coiled coils. (O'Shea et al.,Science 254:539; 1991) The general architecture of the parallel coiledcoil has been well characterized, with a “knobs-into-holes” packing asproposed by Crick in 1953 (Acta Crystallogr. 6:689). The dimer formed bya zipper domain is stabilized by the heptad repeat, designated(abcdefg)_(n) according to the notation of McLachlan and Stewart (J.Mol. Biol. 98:293; 1975), in which residues a and d are generallyhydrophobic residues, with d being a leucine, which line up on the sameface of a helix. Oppositely-charged residues commonly occur at positionsg and e. Thus, in a parallel coiled coil formed from two helical zipperdomains, the “knobs” formed by the hydrophobic side chains of the firsthelix are packed into the “holes” formed between the side chains of thesecond helix.

The residues at position d (often leucine) contribute large hydrophobicstabilization energies, and are important for oligomer formation(Krystek: et al., Int. J. Peptide Res. 38:229, 1991). Lovejoy et al.(Science 259:1288, 1993) recently reported the synthesis of atriple-stranded α-helical bundle in which the helices run up-up-down.Their studies confirmed that hydrophobic stabilization energy providesthe main driving force for the formation of coiled coils from helicalmonomers. These studies also indicate that electrostatic interactionscontribute to the stoichiometry and geometry of coiled coils. Furtherdiscussion of the structure of leucine zippers is found in Harbury etal. (Science 262:1401, 26 Nov. 1993)

Examples of leucine zipper domains suitable for producing solubleoligomeric proteins are described in PCT application WO 94/10308, andthe leucine zipper derived from lung surfactant protein D (SPD)described in Hoppe et al. (FEBS Letters 344:191, 1994), herebyincorporated by reference. The use of a modified leucine zipper thatallows for stable trimerization of a heterologous protein fused theretois described in Fanslow et al. (Semin. Immunol. 6:267–278, 1994).Recombinant fusion proteins comprising a soluble polypeptide fused to aleucine zipper peptide are expressed in suitable host cells, and thesoluble oligomer that forms is recovered from the culture supernatant.

Certain leucine zipper moieties preferentially form trimers. One exampleis a leucine zipper derived from lung surfactant protein D (SPD), asdescribed in Hoppe et al. (FEBS Letters 344:191, 1994) and in U.S. Pat.No. 5,716,805, hereby incorporated by reference in their entirety. Thislung SPD-derived leucine zipper peptide comprises the amino acidsequence Pro Asp Val Ala Ser Leu Arg Gln Gln Val Glu Ala Leu Gln Gly GlnVal Gln His Leu Gln Ala Ala Phe Ser Gln Tyr (SEQ ID NO:4).

Another example of a leucine zipper that promotes trimerization is apeptide comprising the amino acid sequence Arg Met Lys Gln Ile Glu AspLys Ile Glu Glu Ile Leu Ser Lys Ile Tyr His Ile Glu Asn Glu Ile Ala ArgIle Lys Lys Leu Ile Gly Glu Arg, as described in U.S. Pat. No. 5,716,805(SEQ ID NO:5). In one alternative embodiment, an N-terminal Asp residueis added; in another, the peptide lacks the N-terminal Arg residue.

Fragments of the foregoing zipper peptides that retain the property ofpromoting oligomerization may be employed as well. Examples of suchfragments include, but are not limited to, peptides lacking one or twoof the N-terminal or C-terminal residues presented in the foregoingamino acid sequences. Leucine zippers may be derived from naturallyoccurring leucine zipper peptides, e.g., via conservativesubstitution(s) in the native amino acid sequence, wherein the peptide'sability to promote oligomerization is retained.

Other peptides derived from naturally occurring trimeric proteins may beemployed in preparing trimeric oligomers. Alternatively, syntheticpeptides that promote oligomerization may be employed. In particularembodiments, leucine residues in a leucine zipper moiety are replaced byisoleucine residues. Such peptides comprising isoleucine may be referredto as isoleucine zippers, but are encompassed by the term “leucinezippers” as employed herein.

Production of Polypeptides and Fragments Thereof

Expression, isolation and purification of the polypeptides and fragmentsof the invention may be accomplished by any suitable technique,including but not limited to the following:

Expression Systems

The present invention also provides recombinant cloning and expressionvectors containing DNA, as well as host cell containing the recombinantvectors. Expression vectors comprising DNA may be used to prepare thepolypeptides or fragments of the invention encoded by the DNA. A methodfor producing polypeptides comprises culturing host cells transformedwith a recombinant expression vector encoding the polypeptide, underconditions that promote expression of the polypeptide, then recoveringthe expressed polypeptides from the culture. The skilled artisan willrecognize that the procedure for purifying the expressed polypeptideswill vary according to such factors as the type of host cells employed,and whether the polypeptide is membrane-bound or a soluble form that issecreted from the host cell.

Any suitable expression system may be employed. The vectors include aDNA encoding a polypeptide or fragment of the invention, operably linkedto suitable transcriptional or translational regulatory nucleotidesequences, such as those derived from a mammalian, microbial, viral, orinsect gene. Examples of regulatory sequences include transcriptionalpromoters, operators, or enhancers, an mRNA ribosomal binding site, andappropriate sequences which control transcription and translationinitiation and termination. Nucleotide sequences are operably linkedwhen the regulatory sequence functionally relates to the DNA sequence.Thus, a promoter nucleotide sequence is operably linked to a DNAsequence if the promoter nucleotide sequence controls the transcriptionof the DNA sequence. An origin of replication that confers the abilityto replicate in the desired host cells, and a selection gene by whichtransformants are identified, are generally incorporated into theexpression vector.

In addition, a sequence encoding an appropriate signal peptide (nativeor heterologous) can be incorporated into expression vectors. A DNAsequence for a signal peptide (secretory leader) may be fused in frameto the polynucleotide sequence of the invention so that the DNA isinitially transcribed, and the mRNA translated, into a fusion proteincomprising the signal peptide. A signal peptide that is functional inthe intended host cells promotes extracellular secretion of thepolypeptide. The signal peptide is cleaved from the polypeptide uponsecretion of polypeptide from the cell.

The skilled artisan will also recognize that the position(s) at whichthe signal peptide is cleaved may differ from that predicted by computerprogram, and may vary according to such factors as the type of hostcells employed in expressing a recombinant polypeptide. A proteinpreparation may include a mixture of protein molecules having differentN-terminal amino acids, resulting from cleavage of the signal peptide atmore than one site.

Suitable host cells for expression of polypeptides include prokaryotes,yeast or higher eukaryotic cells. Mammalian or insect cells aregenerally preferred for use as host cells. Appropriate cloning andexpression vectors for use with bacterial, fungal, yeast, and mammaliancellular hosts are described, for example, in Pouwels et al. CloningVectors: A Laboratory Manual, Elsevier, New York, (1985). Cell-freetranslation systems could also be employed to produce polypeptides usingRNAs derived from DNA constructs disclosed herein.

Prokaryotic Systems

Prokaryotes include gram-negative or gram-positive organisms. Suitableprokaryotic host cells for transformation include, for example, E. coli,Bacillus subtilis, Salmonella typhimurium, and various other specieswithin the genera Pseudomonas, Streptomyces, and Staphylococcus. In aprokaryotic host cell, such as E. coli, a polypeptide may include anN-terminal methionine residue to facilitate expression of therecombinant polypeptide in the prokaryotic host cell. The N-terminal Metmay be cleaved from the expressed recombinant polypeptide.

Expression vectors for use in prokaryotic host cells generally compriseone or more phenotypic selectable marker genes. A phenotypic selectablemarker gene is, for example, a gene encoding a protein that confersantibiotic resistance or that supplies an autotrophic requirement.Examples of useful expression vectors for prokaryotic host cells includethose derived from commercially available plasmids such as the cloningvector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin andtetracycline resistance and thus provides simple means for identifyingtransformed cells. An appropriate promoter and a DNA sequence areinserted into the pBR322 vector. Other commercially available vectorsinclude, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden) and pGEM1 (Promega Biotec, Madison, Wis., USA).

Promoter sequences commonly used for recombinant prokaryotic host cellexpression vectors include β-lactamase (penicillinase), lactose promotersystem (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature281:544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl.Acids Res. 8:4057, 1980; and EP-A-36776) and tac promoter (Maniatis,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,p. 412, 1982). A particularly useful prokaryotic host cell expressionsystem employs a phage λP_(L) promoter and a cI857ts thermolabilerepressor sequence. Plasmid vectors available from the American TypeCulture Collection which incorporate derivatives of the λP_(L) promoterinclude plasmid pHUB2 (resident in E. coli strain JMB9, ATCC 37092) andpPLc28 (resident in E. coli RR1, ATCC 53082).

Yeast Systems

Alternatively, the polypeptides may be expressed in yeast host cells,preferably from the Saccharomyces genus (e.g., S. cerevisiae). Othergenera of yeast, such as Pichia or Kluyveromyces, may also be employed.Yeast vectors will often contain an origin of replication sequence froma 2μ yeast plasmid, an autonomously replicating sequence (ARS), apromoter region, sequences for polyadenylation, sequences fortranscription termination, and a selectable marker gene. Suitablepromoter sequences for yeast vectors include, among others, promotersfor metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J.Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J.Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900,1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phospho-glucose isomerase, andglucokinase. Other suitable vectors and promoters for use in yeastexpression are further described in Hitzeman, EPA-73,657. Anotheralternative is the glucose-repressible ADH2 promoter described byRussell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al. (Nature300:724, 1982). Shuttle vectors replicable in both yeast and E. coli maybe constructed by inserting DNA sequences from pBR322 for selection andreplication in E. coli (Amp^(r) gene and origin of replication) into theabove-described yeast vectors.

The yeast α-factor leader sequence may be employed to direct secretionof the polypeptide. The α-factor leader sequence is often insertedbetween the promoter sequence and the structural gene sequence. See,e.g., Kurjan et al., Cell 30:933, 1982 and Bitter et al., Proc. Natl.Acad. Sci. USA 81:5330, 1984. Other leader sequences suitable forfacilitating secretion of recombinant polypeptides from yeast hosts areknown to those of skill in the art. A leader sequence may be modifiednear its 3′ end to contain one or more restriction sites. This willfacilitate fusion of the leader sequence to the structural gene.

Yeast transformation protocols are known to those of skill in the art.One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci.USA 75:1929, 1978. The Hinnen et al. protocol selects for Trp⁺transformants in a selective medium, wherein the selective mediumconsists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose,10 mg/ml adenine and 20 mg/ml uracil.

Yeast host cells transformed by vectors containing an ADH2 promotersequence may be grown for inducing expression in a “rich” medium. Anexample of a rich medium is one consisting of 1% yeast extract, 2%peptone, and 1% glucose supplemented with 80 mg/ml adenine and 80 mg/mluracil. Derepression of the ADH2 promoter occurs when glucose isexhausted from the medium.

Mammalian or Insect Systems

Mammalian or insect host cell culture systems also may be employed toexpress recombinant polypeptides. Bacculovirus systems for production ofheterologous proteins in insect cells are reviewed by Luckow andSummers, Bio/Technology 6:47 (1988). Established cell lines of mammalianorigin also may be employed. Examples of suitable mammalian host celllines include the COS-7 line of monkey kidney cells (ATCC CRL 1651)(Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3 cells(ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK(ATCC CRL 10) cell lines, and the CV1/EBNA cell line derived from theAfrican green monkey kidney cell line CV1 (ATCC CCL 70) as described byMcMahan et al. (EMBO J. 10: 2821, 1991).

Established methods for introducing DNA into mammalian cells have beendescribed (Kaufman, R. J., Large Scale Mammalian Cell Culture, 1990, pp.15–69). Additional protocols using commercially available reagents, suchas Lipofectamine lipid reagent (Gibco/BRL) or Lipofectamine-Plus lipidreagent, can be used to transfect cells (Feigner et al., Proc. Natl.Acad. Sci. USA 84:7413–7417, 1987). In addition, electroporation can beused to transfect mammalian cells using conventional procedures, such asthose in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2 ed.Vol. 1–3, Cold Spring Harbor Laboratory Press, 1989). Selection ofstable transformants can be performed using methods known in the art,such as, for example, resistance to cytotoxic drugs. Kaufman et al.,Meth. in Enzymology 185:487–511, 1990, describes several selectionschemes, such as dihydrofolate reductase (DHFR) resistance. A suitablehost strain for DHFR selection can be CHO strain DX-B11, which isdeficient in DHFR (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA77:4216–4220, 1980). A plasmid expressing the DHFR cDNA can beintroduced into strain DX-B11, and only cells that contain the plasmidcan grow in the appropriate selective media. Other examples ofselectable markers that can be incorporated into an expression vectorinclude cDNAs conferring resistance to antibiotics, such as G418 andhygromycin B. Cells harboring the vector can be selected on the basis ofresistance to these compounds.

Transcriptional and translational control sequences for mammalian hostcell expression vectors can be excised from viral genomes. Commonly usedpromoter sequences and enhancer sequences are derived from polyomavirus, adenovirus 2, simian virus 40 (SV40), and human cytomegalovirus.DNA sequences derived from the SV40 viral genome, for example, SV40origin, early and late promoter, enhancer, splice, and polyadenylationsites can be used to provide other genetic elements for expression of astructural gene sequence in a mammalian host cell. Viral early and latepromoters are particularly useful because both are easily obtained froma viral genome as a fragment, which can also contain a viral origin ofreplication (Fiers et al., Nature 273:113, 1978; Kaufman, Meth. inEnzymology, 1990). Smaller or larger SV40 fragments can also be used,provided the approximately 250 bp sequence extending from the Hind IIIsite toward the Bgl I site located in the SV40 viral origin ofreplication site is included.

Additional control sequences shown to improve expression of heterologousgenes from mammalian expression vectors include such elements as theexpression augmenting sequence element (EASE) derived from CHO cells(Morris et al., Animal Cell Technology, 1997, pp. 529–534 and PCTApplication WO 97/25420) and the tripartite leader (TPL) and VA geneRNAs from Adenovirus 2 (Gingeras et al., J. Biol. Chem. 257:13475–13491,1982). The internal ribosome entry site (IRES) sequences of viral originallows dicistronic mRNAs to be translated efficiently (Oh and Sarnow,Current Opinion in Genetics and Development 3:295–300, 1993; Ramesh etal., Polynucleotides Research 24:2697–2700, 1996). Expression of aheterologous cDNA as part of a dicistronic mRNA followed by the gene fora selectable marker (e.g. DHFR) has been shown to improvetransfectability of the host and expression of the heterologous cDNA(Kaufman, Meth. in Enzymology, 1990). Exemplary expression vectors thatemploy dicistronic mRNAs are pTR-DC/GFP described by Mosser et al.,Biotechniques 22:150–161, 1997, and p2A5I described by Morris et al.,Animal Cell Technology, 1997, pp. 529–534.

A useful high expression vector, pCAVNOT, has been described by Mosleyet al., Cell 59:335–348, 1989. Other expression vectors for use inmammalian host cells can be constructed as disclosed by Okayama and Berg(Mol. Cell. Biol. 3:280, 1983). A useful system for stable high levelexpression of mammalian cDNAs in C127 murine mammary epithelial cellscan be constructed substantially as described by Cosman et al. (Mol.Immunol. 23:935, 1986). A useful high expression vector, PMLSV N1/N4,described by Cosman et al., Nature 312:768, 1984, has been deposited asATCC 39890. Additional useful mammalian expression vectors are describedin EP-A-0367566, and in WO 91/18982, incorporated by reference herein.In yet another alternative, the vectors can be derived fromretroviruses.

Another useful expression vector, pFLAG®, can be used. FLAG® technologyis centered on the fusion of a low molecular weight (1 kD), hydrophilic,FLAG® marker peptide to the N-terminus of a recombinant proteinexpressed by pFLAG® expression vectors.

Regarding signal peptides that may be employed, the native signalpeptide may be replaced by a heterologous signal peptide or leadersequence, if desired. The choice of signal peptide or leader may dependon factors such as the type of host cells in which the recombinantpolypeptide is to be produced. To illustrate, examples of heterologoussignal peptides that are functional in mammalian host cells include thesignal sequence for interleukin-7 (IL-7) described in U.S. Pat. No.4,965,195; the signal sequence for interleukin-2 receptor described inCosman et al., Nature 312:768 (1984); the interleukin-4 receptor signalpeptide described in EP 367,566; the type I interleukin-1 receptorsignal peptide described in U.S. Pat. No. 4,968,607; and the type IIinterleukin-1 receptor signal peptide described in EP 460,846.

Isolation and Purification

The “isolated” polypeptides or fragments thereof encompassed by thisinvention are polypeptides or fragments that are not in an environmentidentical to an environment in which it or they can be found in nature.The “purified” polypeptides or fragments thereof encompassed by thisinvention are essentially free of association with other proteins orpolypeptides, for example, as a purification product of recombinantexpression systems such as those described above or as a purifiedproduct from a non-recombinant source such as naturally occurring cellsand/or tissues.

In one preferred embodiment, the purification of recombinantpolypeptides or fragments can be accomplished using fusions ofpolypeptides or fragments of the invention to another polypeptide to aidin the purification of polypeptides or fragments of the invention. Suchfusion partners can include the poly-His or other antigenicidentification peptides described above as well as the Fc moietiesdescribed previously.

With respect to any type of host cell, as is known to the skilledartisan, procedures for purifying a recombinant polypeptide or fragmentwill vary according to such factors as the type of host cells employedand whether or not the recombinant polypeptide or fragment is secretedinto the culture medium.

In general, the recombinant polypeptide or fragment can be isolated fromthe host cells if not secreted, or from the medium or supernatant ifsoluble and secreted, followed by one or more concentration,salting-out, ion exchange, hydrophobic interaction, affinitypurification or size exclusion chromatography steps. As to specific waysto accomplish these steps, the culture medium first can be concentratedusing a commercially available protein concentration filter, forexample, an Amicon or Millipore Pellicon ultrafiltration unit. Followingthe concentration step, the concentrate can be applied to a purificationmatrix such as a gel filtration medium. Alternatively, an anion exchangeresin can be employed, for example, a matrix or substrate having pendantdiethylaminoethyl (DEAE) groups. The matrices can be acrylamide,agarose, dextran, cellulose or other types commonly employed in proteinpurification. Alternatively, a cation exchange step can be employed.Suitable cation exchangers include various insoluble matrices comprisingsulfopropyl or carboxymethyl groups. In addition, a chromatofocusingstep can be employed. Alternatively, a hydrophobic interactionchromatography step can be employed. Suitable matrices can be phenyl oroctyl moieties bound to resins. In addition, affinity chromatographywith a matrix which selectively binds the recombinant protein can beemployed. Examples of such resins employed are lectin columns, dyecolumns, and metal-chelating columns. Finally, one or more reverse-phasehigh performance liquid chromatography (RP-HPLC) steps employinghydrophobic RP-HPLC media, (e.g., silica gel or polymer resin havingpendant methyl, octyl, octyldecyl or other aliphatic groups) can beemployed to further purify the polypeptides. Some or all of theforegoing purification steps, in various combinations, are well knownand can be employed to provide an isolated and purified recombinantprotein.

It is also possible to utilize an affinity column comprising apolypeptide-binding protein of the invention, such as a monoclonalantibody generated against polypeptides of the invention, toaffinity-purify expressed polypeptides. These polypeptides can beremoved from an affinity column using conventional techniques, e.g., ina high salt elution buffer and then dialyzed into a lower salt bufferfor use or by changing pH or other components depending on the affinitymatrix utilized, or be competitively removed using the naturallyoccurring substrate of the affinity moiety, such as a polypeptidederived from the invention.

In this aspect of the invention, polypeptide-binding proteins, such asthe anti-polypeptide antibodies of the invention or other proteins thatmay interact with the polypeptide of the invention, can be bound to asolid phase support such as a column chromatography matrix or a similarsubstrate suitable for identifying, separating, or purifying cells thatexpress polypeptides of the invention on their surface. Adherence ofpolypeptide-binding proteins of the invention to a solid phasecontacting surface can be accomplished by any means. For example,magnetic microspheres can be coated with these polypeptide-bindingproteins and held in the incubation vessel through a magnetic field.Suspensions of cell mixtures are contacted with the solid phase that hassuch polypeptide-binding proteins thereon. Cells having polypeptides ofthe invention on their surface bind to the fixed polypeptide-bindingprotein and unbound cells then are washed away. This affinity-bindingmethod is useful for purifying, screening, or separating suchpolypeptide-expressing cells from solution. Methods of releasingpositively selected cells from the solid phase are known in the art andencompass, for example, the use of enzymes. Such enzymes are preferablynon-toxic and non-injurious to the cells and are preferably directed tocleaving the cell-surface binding partner.

Alternatively, mixtures of cells suspected of containingpolypeptide-expressing cells of the invention first are incubated with abiotinylated polypeptide-binding protein of the invention. Incubationperiods are typically at least one hour in duration to ensure sufficientbinding to polypeptides of the invention. The resulting mixture then ispassed through a column packed with avidin-coated beads, whereby thehigh affinity of biotin for avidin provides the binding of thepolypeptide-binding cells to the beads. Use of avidin-coated beads isknown in the art. See Berenson, et al. J. Cell. Biochem., 10D:239(1986). Wash of unbound material and the release of the bound cells isperformed using conventional methods.

The desired degree of purity depends on the intended use of the protein.A relatively high degree of purity is desired when the polypeptide is tobe administered in vivo, for example. In such a case, the polypeptidesare purified such that no protein bands corresponding to other proteinsare detectable upon analysis by SDS-polyacrylamide gel electrophoresis(SDS-PAGE). It will be recognized by one skilled in the pertinent fieldthat multiple bands corresponding to the polypeptide may be visualizedby SDS-PAGE, due to differential glycosylation, differentialpost-translational processing, and the like. Most preferably, thepolypeptide of the invention is purified to substantial homogeneity, asindicated by a single protein band upon analysis by SDS-PAGE. Theprotein band may be visualized by silver staining, Coomassie bluestaining, or (if the protein is radiolabeled) by autoradiography.

Screening Assays

The purified polypeptides of the invention (including proteins,polypeptides, fragments, variants, oligomers, and other forms) may betested for the ability to bind the binding partner in any suitableassay, such as a conventional binding assay. The polypeptide may belabeled with a detectable reagent (e.g., a radionuclide, chromophore,enzyme that catalyzes a colorimetric or fluorometric reaction, and thelike). The labeled polypeptide is contacted with cells expressing thebinding partner. The cells then are washed to remove unbound labeledpolypeptide, and the presence of cell-bound label is determined by asuitable technique, chosen according to the nature of the label.

Another type of suitable binding assay is a competitive binding assay.Competitive binding assays can be performed by conventional methodology.Reagents that may be employed in competitive binding assays includeradiolabeled polypeptides of the invention and intact cells expressingthe binding partner (endogenous or recombinant). For example, aradiolabeled soluble IL-1 eta fragment can be used to compete with asoluble IL-1 eta variant for binding to cell surface IL-1 eta receptors.Instead of intact cells, one could substitute a soluble bindingpartner/Fc fusion protein bound to a solid phase through the interactionof Protein A or Protein G (on the solid phase) with the Fc moiety.Chromatography columns that contain Protein A and Protein G includethose available from Pharmacia Biotech, Inc., Piscataway, N.J.

Another type of competitive binding assay utilizes radiolabeled solublebinding partner, such as a soluble IL-1 eta receptor/Fc fusion protein,and intact cells expressing the binding partner. Qualitative results canbe obtained by competitive autoradiographic plate binding assays, whileScatchard plots (Scatchard, Ann. N.Y. Acad. Sci. 51:660, 1949) may beutilized to generate quantitative results. Such binding assays may beuseful in evaluating the biological activity of a variant polypeptide byassaying for the variant's ability to compete with the native proteinfor binding to the binding partner.

The IL-1 eta polypeptide of the present invention may also be used in ascreening assay for compounds and small molecules which inhibitactivation by (antagonize) the IL-1 eta polypeptide of the instantinvention. Thus, polypeptides of the invention may be used to identifyantagonists from, for example, cells, cell-free preparations, chemicallibraries, and natural product mixtures. The antagonists may be naturalor modified substrates, ligands, enzymes, receptors, etc. of the IL-1eta polypeptide, or may be structural or functional mimetics of the IL-1eta polypeptide. The antagonists may further be small molecules,peptides, antibodies and antisense oligonucleotides.

One embodiment of a method for identifying compounds which antagonizethe IL-1 eta polypeptide is contacting a candidate compound with cellswhich respond to IL-1 eta polypeptide and observe the binding of IL-1eta to the cells, or stimulation or inhibition of a functional response.The activity of the cells which were contacted with the candidatecompound could then be compared with the identical cells which were notcontacted for IL-1 eta polypeptide activity and IL-1 eta polypeptideagonists and antagonists could be identified. A still further embodimentof the instant invention provides a method of identifying compounds thatinhibit the synthesis or secretion of IL-1 eta by contacting thecandidate compound with cells which express IL-1 eta polypeptide andmeasuring the IL-1 eta production. The measurement of IL-1 etaproduction could be performed by a number of well-known methods such asmeasuring the amount of protein present (e.g. an ELISA) or of theprotein's activity.

Drug Discovery

The purified polypeptides according to the invention will facilitate thediscovery of inhibitors (or antagonists) and/or agonists of suchpolypeptides. The use of a purified polypeptide of the invention in thescreening of potential inhibitors and/or agonists thereof is importantand can eliminate or reduce the possibility of interfering reactionswith contaminants.

In addition, polypeptides of the invention can be used forstructure-based design of polypeptide-inhibitors and/or agonists. Suchstructure-based design is also known as “rational drug design.” Thepolypeptides can be three-dimensionally analyzed by, for example, X-raycrystallography, nuclear magnetic resonance or homology modeling, all ofwhich are well-known methods. The use of the polypeptide structuralinformation in molecular modeling software systems to assist ininhibitor design and inhibitor-polypeptide interaction is alsoencompassed by the invention. Such computer-assisted modeling and drugdesign can utilize information such as chemical conformational analysis,electrostatic potential of the molecules, protein folding, etc. Forexample, most of the design of class-specific inhibitors ofmetalloproteases has focused on attempts to chelate or bind thecatalytic zinc atom. Synthetic inhibitors are usually designed tocontain a negatively-charged moiety to which is attached a series ofother groups designed to fit the specificity pockets of the particularprotease. A particular method of the invention comprises analyzing thethree dimensional structure of polypeptides of the invention for likelybinding sites of substrates, synthesizing a new molecule thatincorporates a predictive reactive site, and assaying the new moleculeas described above.

Specific screening methods are known in the art and along withintegrated robotic systems and collections of chemical compounds/naturalproducts are extensively incorporated in high throughput screening sothat large numbers of test compounds can be tested for antagonist oragonist activity within a short amount of time. These methods includehomogeneous assay formats such as fluorescence resonance energytransfer, fluorescence polarization, time-resolved fluorescenceresonance energy transfer, scintillation proximity assays, reporter geneassays, fluorescence quenched enzyme substrate, chromogenic enzymesubstrate and electrochemiluminescence, as well as more traditionalheterogeneous assay formats such as enzyme-linked immunosorbant assays(ELISA) or radioimmunoassays. Homogeneous assays are preferred. Alsocomprehended herein are cell-based assays, for example those utilizingreporter genes, as well as functional assays that analyze the effect ofan antagonist or agonist on biological function(s) or activity(ies) ofIL-1 eta (for example, stimulation of the secretion of cytokines orinhibition thereof, as disclosed herein). Moreover, animal models ofinflammatory conditions are useful assays of biological activity.

Accordingly, in one aspect of the invention, there is provided a methodfor screening a test compound to determine whether the test compoundaffects (or modulates) a biological activity of an IL-1 eta polypeptide,the method comprising contacting the test compound and the IL-1 etapolypeptide with cells capable of exhibiting the biological activitywhen contacted with IL-1 eta, and analyzing the cells for the occurrenceof the biological activity, wherein if the biological activity observedin the presence of the test compound differs from the biologicalactivity that is observed when the test compound is absent, the testcompound affects the biological activity of the IL-1 eta. The cells maybe contacted in vitro or in vivo.

As used herein, the IL-1 eta polypeptide comprises a polypeptideselected from the group consisting of the polypeptides of SEQ ID NO:2,and polypeptides encoded by DNAs that hybridize under moderatelystringent conditions to the DNA of SEQ ID NO:1. Such polypeptidesinclude polypeptides comprising variant amino acid sequences that are atleast 80% identical to the polypeptides of SEQ ID NO:2 (preferably, thevariant amino acid sequences that are at least 90% identical, morepreferably at least 95% identical, most preferably at least 97%identical, to the polypeptides of SEQ ID NO:2). Additional examples ofuseful IL-1 eta polypeptides include polypeptides comprising the aminoacid sequences of SEQ ID NO:2 wherein the polypeptides comprisealterations to the amino acid sequences selected from the groupconsisting of inactivated N-glycosylation site(s), inactivated proteaseprocessing site(s), conservative amino acid substitution(s), andcombinations thereof. Moreover, fragments of the aforesaid polypeptidesthat have at least one activity of IL-1 eta as described below are alsocomprehended herein.

IL-1 eta biological activity includes, but is not limited to, modulationof cytokine expression, modulation of the expression of moleculesindicative of activation of an immune or inflammatory response (forexample, COX2, iNOS), modulation of cell-surface molecule expression,modulation of activation of one or more signaling cascades, modulationof induction of mRNAs for the aforementioned proteins, modulation ofinduction of cell proliferation and/or cell death, induction ofmorphological and/or functional changes in cells, and combinationsthereof. The inventive methods comprise methods of assaying for any ofthese biological activities. Those of skill in the art will recognizethat modulation of cytokines means that the levels of expression ofcertain cytokines increase while the levels of other cytokinesdecreases, and that such combinations are comprehended in the termmodulation; the same is true for other activities of IL-1 eta.

When the methods of the present invention include assaying for IL-1 etamodulation of cytokine expression, cytokines that may be assayed include(but are not limited to) IL-1 alpha, IL-1 beta, TNF-alpha, IL-10,IFN-gamma, IL-12 (in particular, the p40 subunit), IL-6, IL-1ra, IL-4,IL-13, GM-CSF, IL-18, IL-1 homologs such as IL-1 epsilon, IL-1 eta, IL-1theta, IL-1 zeta, and IL-1 H1, and combinations thereof. Similarly, whenthe screening methods of the present invention include assaying for IL-1eta modulation of cell surface molecule expression, the cell surfacemolecules that may be assayed include ICAM-1, TLR4, TLR5, TLR9, DC-B7,MHC class I and II antigens, VCAM, ELAM, B7-1, B7-2, CD40L, andcombinations thereof.

IL-1 eta mediated modulation of signaling pathways often involves acascade of molecular changes, for example as discussed previouslywherein a receptor propagates a ligand-receptor mediated signal byspecifically activating intracellular kinases which phosphorylate targetsubstrates (which can themselves be kinases that become activatedfollowing phosphorylation, or adaptor molecules that facilitatedown-stream signaling through protein-protein interaction followingphosphorylation), resulting in the activation of other factors (forexample, NFkappaB). When the screening methods of the present inventioninclude assaying for IL-1 eta induced modulation of signaling pathways,the signaling pathways that may be assayed include those involvingactivation of NFkappaB. Assaying for activation signaling cascadesfurther includes detecting phosphorylation of molecules that occursduring the signaling cascade, as in the phosphorylation of IkappaB(including IkappaB degradation assays, and assays for free IkappaB), p38MAP kinase, and Stress-Activated Protein Kinase (SAPK/JNK).

Moreover, those of skill in the art understand that biologicalactivity(ies) is/are most often induced by the binding of a ligand(i.e., IL-1 eta) to a receptor (counterstructure or binding moiety)present on a cell; accordingly, as previously described, IL-1 etapolypeptides (including IL-1 eta polypeptide fragments) can be used inbinding studies to identify receptor-expressing cells. Such bindingstudies also provide assays useful in the inventive methods. IL-1 etapolypeptides may also be used to clone receptors (or other moleculesthat bind IL-1 eta) and to screen for molecules that blockreceptor/ligand interactions. Those of ordinary skill in the art furtherunderstand that biological activities include cell proliferation, celldeath, and changes in cell morphology and/or function (for example,activation, maturation); assays that evaluate such effects of IL-1 etaare known in the art, and will also be useful in the inventive methods.Moreover, animal models of syndromes and/or conditions, such as thosedisclosed herein, are useful for screening compounds for biologicalactivity, including screening for antagonism (or agonism) of IL-1 eta.

The inventive methods further encompass performing more than one assayto discover and/or analyze agonists or antagonists of IL-1 eta activity(i.e., combination methods). Generally, such methods comprise selectingtest compounds that affect a property of IL-1 eta (i.e., an ability ofIL-1 eta to bind an IL-1 eta counter structure), then testing theselected compounds for an effect on another property of IL-1 eta (i.e.,contacting the selected test compounds and an IL-1 eta polypeptide withcells capable of exhibiting a biological activity when contacted withIL-1 eta, and determining whether the compounds affect the biologicalactivity). For example, the inventive methods may comprise a first assayto determine whether a candidate molecule interacts with (binds to) IL-1eta. In one embodiment, the first assay is in a high throughput format,numerous forms of which are known in the art and disclosed herein. Suchan assay will generally comprise the steps of: contacting test compoundsand an IL-1 eta polypeptide with an IL-1 eta counterstructure;determining whether the test compounds affect the ability of IL-1 eta tobind the counterstructure; and selecting one or more test compounds thataffect the ability of IL-1 eta to bind the counterstructure. Theinventive combination methods further comprise evaluating selectedcompounds in a second assay, for agonistic or antagonistic effect onbiological activity using one or more of the aforementioned assays.

Alternatively, the inventive combination methods may comprise a firstassay to determine whether a candidate molecule modulates a biologicalactivity of IL-1 eta, as described herein using an in vitro assay or anin vivo assay (for example, an animal model). According to suchcombination methods, molecules that modulate an IL-1 eta biologicalactivity in this manner are selected using one or more of theaforementioned assays for biological activity, and assayed to determinewhether the candidate molecule(s) bind IL-1 eta. The selected moleculesmay be tested to further define the exact region or regions of IL-1 etato which the test molecule binds (for example, epitope mapping forantibodies).

As disclosed previously, the types of assays for biological activitiesof IL-1 eta that can be used in the inventive combination methodsinclude assays for the expression of cytokines, assays for theexpression of cell-surface molecules, assays to detect activation ofsignaling molecules, assays to detect induction of mRNAs, and assaysthat evaluate cell proliferation or cell death (and combinationsthereof), as described herein. Molecules that bind and that have anagonistic or antagonistic effect on biologic activity will be useful intreating or preventing diseases or conditions with which thepolypeptide(s) are implicated.

Those of ordinary skill in the art understand that when the biologicalactivity observed in the presence of the test compound is greater thanthat observed when the test compound is absent, the test compound is anagonist of IL-1 eta, whereas when the biological activity observed inthe presence of the test compound is less than that observed when thetest compound is absent, the test compound is an antagonist (orinhibitor) of IL-1 eta. Generally, an antagonist will decrease orinhibit, an activity by at least 30%; more preferably, antagonists willinhibit activity by at least 50%, most preferably by at least 90%.Similarly, an agonist will increase, or enhance, an activity by at least20%; more preferably, agonists will enhance activity by at least 30%,most preferably by at least 50%. Those of skill in the art will alsorecognize that agonists and/or antagonists with different levels ofagonism or antagonism respectively may be useful for differentapplications (i.e., for treatment of different disease states).

Homogeneous assays are mix-and-read style assays that are very amenableto robotic application, whereas heterogeneous assays require separationof free from bound analyte by more complex unit operations such asfiltration, centrifugation or washing. These assays are utilized todetect a wide variety of specific biomolecular interactions (includingprotein-protein, receptor-ligand, enzyme-substrate, and so on), and theinhibition thereof by small organic molecules. These assay methods andtechniques are well known in the art (see, e.g., High ThroughputScreening: The Discovery of Bioactive Substances, John P. Devlin (ed.),Marcel Dekker, New York, 1997 ISBN: 0-8247-0067-8). The screening assaysof the present invention are amenable to high throughput screening ofchemical libraries and are suitable for the identification of smallmolecule drug candidates, antibodies, peptides, and other antagonistsand/or agonists, natural or synthetic. Several useful assays aredisclosed in U.S. Ser. No. 09/851,673, filed May 8, 2001 (the relevantdisclosure of which is hereby incorporated by reference).

Candidate Molecules to be Tested:

The methods of the invention may be used to identify antagonists (alsoreferred to as inhibitors) and agonists of IL-1 eta activity from cells,cell-free preparations, chemical libraries, cDNA libraries, recombinantantibody libraries (or libraries comprising subunits of antibodies) andnatural product mixtures. The antagonists and agonists may be natural ormodified substrates, ligands, enzymes, receptors, etc. of thepolypeptides of the instant invention, or may be structural orfunctional mimetics of IL-1 eta or its binding partner/counterstructure.Potential antagonists of the instant invention include small molecules,peptides and antibodies that bind to and occupy a binding site of theinventive polypeptides or a binding partner thereof, causing them to beunavailable to bind to their natural binding partners and thereforepreventing normal biological activity. Antagonists also includechemicals (including small molecules and peptides) that interfere withthe signaling pathways used by IL-1 eta (for example, by inhibiting theinteraction of receptor subunits, or inhibiting the interaction ofintracellular components of the signaling cascade). Potential agonistsinclude small molecules, peptides and antibodies which bind to theinstant polypeptides or binding partners thereof, and elicit the same orenhanced biologic effects as those caused by the binding of thepolypeptides of the instant invention. Moreover, substances thatactivate (or enhance) the signaling pathways used by IL-1 eta are alsoincluded within the scope of agonists of IL-1 eta.

Small molecule agonists and antagonists are usually less than 10Kmolecular weight and may possess a number of physicochemical andpharmacological properties which enhance cell penetration, resistdegradation and prolong their physiological half-lives (Gibbs, J.,Pharmaceutical Research in Molecular Oncology, Cell, Vol. 79 (1994)).Antibodies, which include intact molecules as well as fragments such asFab and F(ab′)2 fragments, as well as recombinant molecules derivedtherefrom (including antibodies expressed on phage, intrabodies, singlechain antibodies such as scFv and other molecules derived fromimmunoglobulins that are known in the art), may be used to bind to andinhibit the polypeptides of the instant invention by blocking thepropagation of a signaling cascade. It is preferable that the antibodiesare humanized, and more preferable that the antibodies are human. Theantibodies of the present invention may be prepared by any of a varietyof well-known methods, as disclosed herein.

Additional examples of candidate molecules, also referred to herein as“test molecules” or “test compounds,” to be tested for the ability tomodulate IL-1 eta activity include, but are not limited to,carbohydrates, small molecules (usually organic molecules or peptides),proteins, and nucleic acid molecules (including oligonucleotidefragments typically consisting of from 8 to 30 nucleic acid residues).Peptides to be tested typically consist of from 5 to 25 amino acidresidues. Also, candidate nucleic acid molecules can be antisensenucleic acid sequences, and/or can possess ribozyme activity.

Small molecules to be screened using the hereindescribed screeningassays can typically be administered orally or by injection to a patientin need thereof. Small molecules that can be administered orally areespecially preferred. The small molecules of the invention preferablywill not be toxic (or only minimally toxic) at the doses required forthem to be effective as pharmaceutical agents, and they are preferablynot subject to rapid loss of activity in the body, such as the loss ofactivity that might result from rapid enzymatic or chemical degradation.In addition, pharmaceutically useful small molecules are preferably notimmunogenic.

The methods of the invention can be used to screen for antisensemolecules that inhibit the functional expression of one or more mRNAmolecules that encode one or more proteins that mediate an IL-1eta-dependent cellular response. An anti-sense nucleic acid molecule isa DNA sequence that is capable of can hybridizing to the target mRNAmolecule through Watson-Crick base pairing, and inhibiting translationthereof. Alternatively, the DNA may be inverted relative to its normalorientation for transcription and so express an RNA transcript that iscomplementary to the target mRNA molecule (i.e., the RNA transcript ofthe anti-sense nucleic acid molecule can hybridize to the target mRNAmolecule through Watson-Crick base pairing). An anti-sense nucleic acidmolecule may be constructed in a number of different ways provided thatit is capable of interfering with the expression of a target protein.Typical anti-sense oligonucleotides to be screened preferably are 30–40nucleotides in length. The anti-sense nucleic acid molecule generallywill be substantially identical (although in antisense orientation) tothe target gene. The minimal identity will typically be greater thanabout 80%, but a higher identity might exert a more effective repressionof expression of the endogenous sequences. Substantially greateridentity of more than about 90% is preferred, though about 95% toabsolute identity would be most preferred.

Candidate nucleic acid molecules can possess ribozyme activity. Thus,the methods of the invention can be used to screen for ribozymemolecules that inhibit the functional expression of one or more mRNAmolecules that encode one or more proteins that mediate an IL-1 etadependent cellular response. Ribozymes are catalytic RNA molecules thatcan cleave nucleic acid molecules having a sequence that is completelyor partially homologous to the sequence of the ribozyme. It is possibleto design ribozyme transgenes that encode RNA ribozymes thatspecifically pair with a target RNA and cleave the phosphodiesterbackbone at a specific location, thereby functionally inactivating thetarget RNA. In carrying out this cleavage, the ribozyme is not itselfaltered, and is thus capable of recycling and cleaving other molecules.The inclusion of ribozyme sequences within antisense RNAs confersRNA-cleaving activity upon them, thereby increasing the activity of theantisense constructs.

The design and use of target RNA-specific ribozymes is described inHaseloff et al. (Nature, 334:585, 1988; see also U.S. Pat. No.5,646,023), both of which publications are incorporated herein byreference. Tabler et al. (Gene 108:175, 1991) have greatly simplifiedthe construction of catalytic RNAs by combining the advantages of theanti-sense RNA and the ribozyme technologies in a single construct.Smaller regions of homology are required for ribozyme catalysis,therefore this can promote the repression of different members of alarge gene family if the cleavage sites are conserved.

Use of IL-1 eta Polynucleotides and Oligonucleotides

Among the uses of polynucleotides of the invention is the use offragments as probes or primers. Such fragments generally comprise atleast about 17 contiguous nucleotides of a DNA sequence. In otherembodiments, a DNA fragment comprises at least 30, or at least 60,contiguous nucleotides of a DNA sequence.

Because homologs of SEQ ID NO:1, from other mammalian species, arecontemplated herein, probes based on the human DNA sequence of SEQ IDNO:1 may be used to screen cDNA libraries derived from other mammalianspecies, using conventional cross-species hybridization techniques.

Using knowledge of the genetic code in combination with the amino acidsequences set forth above, sets of degenerate oligonucleotides can beprepared. Such oligonucleotides are useful as primers, e.g., inpolymerase chain reactions (PCR), whereby DNA fragments are isolated andamplified.

Polynucleotides encoding SEQ ID NO:2 or oligonucleotide fragments ofsuch polynucleotides, can be used by those skilled in the art usingwell-known techniques to identify the human chromosome 2, as well as thespecific locus thereof, that contains the DNA of IL-1 ligand familymembers. Useful techniques include, but are not limited to, usingpolynucleotides or fragments as probes or primers in techniques thatinclude radiation hybrid mapping (high resolution), in situhybridization to chromosome spreads (moderate resolution), and Southernblot hybridization to hybrid cell lines containing individual humanchromosomes (low resolution).

For example, chromosomes can be mapped by radiation hybridization whichcan include performing PCR amplification using the WhiteheadInstitute/MIT Center for Genome Research Genebridge4 panel of 93radiation hybrids (on the world-wide web (www-) atgenome.wi.mit.edu/ftp/-distribution/human_STS_releases/july97/rhmap/genebridge4.html).Useful PCR primers are those that lie within the gene of interest andwhich amplify a product from human genomic DNA, but do not amplifyhamster genomic DNA. The products of the PCR reactions are convertedinto a data vector that is submitted to the Whitehead/MIT RadiationMapping site on the Internet (on the world-wide web (www-) atseq.wi.mit.edu). The data is scored and the chromosomal assignment andplacement relative to known Sequence Tag Site (STS) markers on theradiation hybrid map is provided. The web site on the world-wide web(www-) at genome.wi.mit.edulftp/distribution/-human_STS₁₃reieases/ju1y97/07-97.INTRO.html) also provides information about radiation hybridmapping.

As set forth below, using radiation hybridization, the polynucleotide ofSEQ ID NO:1 is shown to map to the 2q11-12 region of human chromosome 2.Human chromosome 2 is associated with specific diseases which includebut are not limited to glaucoma, ectodermal dysplasia, insulin-dependentdiabetes mellitus, wrinkly skin syndrome, T-cell leukemia/lymphoma, andtibial muscular dystrophy. Thus, the polynucleotide of SEQ ID NO:1 or afragment thereof can be used by one skilled in the art using well-knowntechniques to analyze abnormalities associated with gene mapping tochromosome 2. This enables one to distinguish conditions in which thismarker is rearranged or deleted. In addition, the polynucleotide of SEQID NO:1 or a fragment thereof can be used as a positional marker to mapother genes of unknown location.

DNA of the present invention may be used in developing treatments forany disorder mediated (directly or indirectly) by defective, orinsufficient amounts of, the genes corresponding to the polynucleotidesof the invention. Disclosure herein of native nucleotide sequencespermits the detection of defective genes, and the replacement thereofwith normal genes. Defective genes may be detected in in vitrodiagnostic assays, and by comparison of a native nucleotide sequencedisclosed herein with that of a gene derived from a person suspected ofharboring a defect in this gene.

Other useful fragments of the polynucleotides of this invention includeantisense or sense oligonucleotides comprising a single-strandedpolynucleotide sequence (either RNA or DNA) capable of binding to targetmRNA (sense) or DNA (antisense) sequences. Antisense or senseoligonucleotides according to the present invention comprise a fragmentof DNA (SEQ ID NO:1). Such a fragment generally comprises at least about14 nucleotides, preferably from about 14 to about 30 nucleotides. Theability to derive an antisense or a sense oligonucleotide, based upon acDNA sequence encoding a given protein is described in, for example,Stein and Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al.(BioTechniques 6:958, 1988).

Binding antisense or sense oligonucleotides to target polynucleotidesequences results in the formation of duplexes that block or inhibitprotein expression by one of several means, including enhanceddegradation of the mRNA by RNAseH, inhibition of splicing, prematuretermination of transcription or translation, or by other means. Theantisense oligonucleotides thus may be used to block expression ofproteins. Antisense or sense oligonucleotides further compriseoligonucleotides having modified sugar-phosphodiester backbones (orother sugar linkages, such as those described in WO91/06629) and whereinsuch sugar linkages are resistant to endogenous nucleases. Sucholigonucleotides with resistant sugar linkages are stable in vivo (i.e.,capable of resisting enzymatic degradation) but retain sequencespecificity to be able to bind to target nucleotide sequences.

Other examples of sense or antisense oligonucleotides include thoseoligonucleotides which are covalently linked to organic moieties, suchas those described in WO 90/10448, and other moieties that increasesaffinity of the oligonucleotide for a target polynucleotide sequence,such as poly-(L-lysine). Further still, intercalating agents, such asellipticine, and alkylating agents or metal complexes may be attached tosense or antisense oligonucleotides to modify binding specificities ofthe antisense or sense oligonucleotide for the target nucleotidesequence.

Antisense or sense oligonucleotides may be introduced into a cellcontaining the target polynucleotide sequence by any gene transfermethod, including, for example, lipofection, CaPO₄-mediated DNAtransfection, electroporation, or by using gene transfer vectors such asEpstein-Barr virus.

Sense or antisense oligonucleotides also may be introduced into a cellcontaining the target nucleotide sequence by formation of a conjugatewith a ligand binding molecule, as described in WO 91/04753. Suitableligand binding molecules include, but are not limited to, cell surfacereceptors, growth factors, other cytokines, or other ligands that bindto cell surface receptors. Preferably, conjugation of the ligand bindingmolecule does not substantially interfere with the ability of the ligandbinding molecule to bind to its corresponding molecule or receptor, orblock entry of the sense or antisense oligonucleotide or its conjugatedversion into the cell.

Alternatively, a sense or an antisense oligonucleotide may be introducedinto a cell containing the target polynucleotide sequence by formationof an oligonucleotide-lipid complex, as described in WO 90/10448. Thesense or antisense oligonucleotide-lipid complex is preferablydissociated within the cell by an endogenous lipase.

Uses of IL-1 eta Polypeptides and Fragmented Polypeptides

Polypeptides of the present invention find use as a protein purificationreagent. The polypeptides may be attached to a solid support materialand used to purify the binding partner proteins by affinitychromatography. In particular embodiments, a polypeptide (in any formdescribed herein that is capable of binding the binding partner) isattached to a solid support by conventional procedures. As one example,chromatography columns containing functional groups that will react withfunctional groups on amino acid side chains of proteins are available(Pharmacia Biotech, Inc., Piscataway, N.J.). In an alternative, apolypeptide/Fc protein (as discussed above) is attached to Protein A- orProtein G-containing chromatography columns through interaction with theFc moiety.

The polypeptide also finds use in purifying or identifying cells thatexpress the binding partner on the cell surface. Polypeptides are boundto a solid phase such as a column chromatography matrix or a similarsuitable substrate. For example, magnetic microspheres can be coatedwith the polypeptides and held in an incubation vessel through amagnetic field. Suspensions of cell mixtures containing the bindingpartner expressing cells are contacted with the solid phase having thepolypeptides thereon. Cells expressing the binding partner on the cellsurface bind to the fixed polypeptides, and unbound cells then arewashed away.

Alternatively, the polypeptides can be conjugated to a detectablemoiety, then incubated with cells to be tested for binding partnerexpression. After incubation, unbound labeled matter is removed and thepresence or absence of the detectable moiety on the cells is determined.

In a further alternative, mixtures of cells suspected of containingcells expressing the binding partner are incubated with biotinylatedpolypeptides. Incubation periods are typically at least one hour induration to ensure sufficient binding. The resulting mixture then ispassed through a column packed with avidin-coated beads, whereby thehigh affinity of biotin for avidin provides binding of the desired cellsto the beads. Procedures for using avidin-coated beads are known (seeBerenson, et al. J. Cell. Biochem., 10D:239, 1986). Washing to removeunbound material, and the release of the bound cells, are performedusing conventional methods.

Polypeptides also find use in measuring the biological activity of thebinding partner protein in terms of their binding affinity. Thepolypeptides thus may be employed by those conducting “qualityassurance” studies, e.g., to monitor shelf life and stability of proteinunder different conditions. For example, the polypeptides may beemployed in a binding affinity study to measure the biological activityof a binding partner protein that has been stored at differenttemperatures, or produced in different cell types. The proteins also maybe used to determine whether biological activity is retained aftermodification of a binding partner protein (e.g., chemical modification,truncation, mutation, etc.). The binding affinity of the modifiedbinding partner protein is compared to that of an unmodified bindingpartner protein to detect any adverse impact of the modifications onbiological activity of the binding partner. The biological activity of abinding partner protein thus can be ascertained before it is used in aresearch study, for example.

The polypeptides also find use as carriers for delivering agentsattached thereto to cells bearing the binding partner. The polypeptidesthus can be used to deliver diagnostic or therapeutic agents to suchcells (or to other cell types found to express the binding partner onthe cell surface) in in vitro or in vivo procedures.

Detectable (diagnostic) and therapeutic agents that may be attached to apolypeptide include, but are not limited to, toxins, other cytotoxicagents, drugs, radionuclides, chromophores, enzymes that catalyze acolorimetric or fluorometric reaction, and the like, with the particularagent being chosen according to the intended application. Among thetoxins are ricin, abrin, diphtheria toxin, Pseudomonas aeruginosaexotoxin A, ribosomal inactivating proteins, mycotoxins such astrichothecenes, and derivatives and fragments (e.g., single chains)thereof. Radionuclides suitable for diagnostic use include, but are notlimited to, ¹²³I, ¹³¹I, ^(99m)Tc, ¹¹¹In, and ⁷⁶Br. Examples ofradionuclides suitable for therapeutic use are ¹³¹I, ²¹¹ At, ⁷⁷Br,¹⁸⁶Re, ¹⁸⁸Re, ²¹²Pb, ²¹²Bi, ¹⁰⁹Pd, ⁶⁴Cu, and ⁶⁷Cu.

Such agents may be attached to the polypeptide by any suitableconventional procedure. The polypeptide comprises functional groups onamino acid side chains that can be reacted with functional groups on adesired agent to form covalent bonds, for example. Alternatively, theprotein or agent may be derivatized to generate or attach a desiredreactive functional group. The derivatization may involve attachment ofone of the bifunctional coupling reagents available for attachingvarious molecules to proteins (Pierce Chemical Company, Rockford, Ill.).A number of techniques for radiolabeling proteins are known.Radionuclide metals may be attached to polypeptides by using a suitablebifunctional chelating agent, for example.

Conjugates comprising polypeptides and a suitable diagnostic ortherapeutic agent (preferably covalently linked) are thus prepared. Theconjugates are administered or otherwise employed in an amountappropriate for the particular application.

Polypeptides of the invention may be used in developing treatments forany disorder mediated (directly or indirectly) by defective, orinsufficient amounts of the polypeptides. Further, the polypeptides ofthe invention may be used in developing treatments for any disorderresulting (directly or indirectly) from an excess of the polypeptide.The polypeptides of the instant invention may be administered to amammal afflicted with such disorders.

The polypeptides may also be employed in inhibiting a biologicalactivity of the binding partner, in in vitro or in vivo procedures. Forexample, a purified IL-1 eta polypeptide can be used to inhibit bindingof endogenous IL-1 eta to its cell surface receptor.

Polypeptides of the invention may be administered to a mammal to treat abinding partner-mediated disorder. Such binding partner-mediateddisorders include conditions caused (directly or indirectly) orexacerbated by the binding partner.

Compositions of the present invention may contain a polypeptide in anyform described herein, such as native proteins, variants, derivatives,oligomers, and biologically active fragments. In particular embodiments,the composition comprises a soluble polypeptide or an oligomercomprising soluble polypeptides of the invention.

Compositions comprising an effective amount of a polypeptide of thepresent invention, in combination with other components such as aphysiologically acceptable diluent, carrier, or excipient, are providedherein. The polypeptides can be formulated according to known methodsused to prepare pharmaceutically useful compositions. They can becombined in admixture, either as the sole active material or with otherknown active materials suitable for a given indication, withpharmaceutically acceptable diluents (e.g., saline, Tris-HCl, acetate,and phosphate buffered solutions), preservatives (e.g., thimerosal,benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/orcarriers. Suitable formulations for pharmaceutical compositions includethose described in Remington's Pharmaceutical Sciences, 16th ed. 1980,Mack Publishing Company, Easton, Pa.

In addition, such compositions can be complexed with polyethylene glycol(PEG), metal ions, or incorporated into polymeric compounds such aspolyacetic acid, polyglycolic acid, hydrogels, dextran, etc., orincorporated into liposomes, microemulsions, micelles, unilamellar ormultilamellar vesicles, erythrocyte ghosts or spheroblasts. Suchcompositions will influence the physical state, solubility, stability,rate of in vivo release, and rate of in vivo clearance, and are thuschosen according to the intended application.

The compositions of the invention can be administered in any suitablemanner, e.g., topically, parenterally, or by inhalation. The term“parenteral” includes injection, e.g., by subcutaneous, intravenous, orintramuscular routes, also including localized administration, e.g., ata site of disease or injury. Those of ordinary skill in the artrecognize that other types of localized administration (e.g.,intraarticular, intracapsular, intracarpal, intracelial,intracerebroventricular, intrasynovial, intraspinal, intraligamentus,intrameningeal, intraocular, epidural, transepithelially, and/oradministration by one or more of these routes at a site near or adjacentto a site of disease or injury) are suitable for use in administeringthe compositions of the present invention. Sustained release fromimplants is also contemplated.

One skilled in the pertinent art will recognize that suitable dosageswill vary, depending upon such factors as the nature of the disorder tobe treated, the patient's body weight, age, and general condition, andthe route of administration. Preliminary doses can be determinedaccording to animal tests, and the scaling of dosages for humanadministration is performed according to art-accepted practices.

Compositions comprising polynucleotides in physiologically acceptableformulations are also contemplated. DNA may be formulated for injection,for example. Moreover, inasmuch as those of ordinary skill in the artare aware that nucleic acid compositions (including DNA) are taken up bycells and result in the expression of protein in or near the area wherethe nucleic acid composition was administered, the inventive nucleicacid compositions will be useful for localized administration ofpolypeptides encoded thereby.

Another use of the polypeptide of the present invention is as a researchtool for studying the biological effects that result from theinteractions of IL-1 eta with its binding partner, or from inhibitingthese interactions, on different cell types. Polypeptides also may beemployed in it vitro assays for detecting IL-1 eta, the binding partneror the interaction thereof. The inventive polypeptides will also beuseful in elucidating the signaling pathways of IL-1 family members, andin identifying molecules that modulate various aspects of such signalingpathways. The modulators identified by studies utilizing the inventivepolypeptides have utility in treating or ameliorating a wide variety ofdiseases and syndromes in which the inflammatory response plays a role.

Another embodiment of the invention relates to uses of the polypeptidesof the invention to study cell signal transduction. IL-1 family ligandsplay a central role in protection against infection and immuneinflammatory responses which includes cellular signal transduction,activating vascular endothelial cells and lymphocytes, induction ofinflammatory cytokines, acute phase proteins, hematopoiesis, fever, boneresorption, prostaglandins, metalloproteinases, and adhesion molecules.With the continued increase in the number of known IL-1 family members,a suitable classification scheme is one based on comparing polypeptidestructure as well as function (activation and regulatory properties).Thus, IL-1 eta, like other IL-1 family ligands (IL-1α, IL-1β, and IL-18)are likely involved in many of the functions noted above as well aspromote inflammatory responses and therefore be involved in thecausation and maintenance of inflammatory and/or autoimmune diseasessuch as rheumatoid arthritis, inflammatory bowel disease, and psoriasis.As such, alterations in the expression and/or activation of thepolypeptides of the invention can have profound effects on a plethora ofcellular processes, including, but not limited to, activation orinhibition of cell specific responses and proliferation. Expression ofcloned IL-1 eta, or of functionally inactive mutants thereof, can beused to identify the role a particular protein plays in mediatingspecific signaling events.

Accordingly, IL-1 eta has therapeutic uses, such as protecting againstinfection and generating immune and inflammatory responses inindividuals whose immune and inflammatory responses are inappropriate ornonresponsive. For example, IL-1 eta may be useful in stimulating theimmune system of individuals whose immune system is immunosuppressed.Similarly, because IL-1 eta likely promotes inflammatory responses andis involved in the causation and maintenance of inflammatory and/orautoimmune diseases, antagonists of IL-1 eta are useful in inhibiting ortreating inflammatory and/or automimmune disease. Thus, antagonists ofIL-1 eta will be useful in treating inflammatory bowel disease (forexample, Crohn's disease and ulcerative colitis), multiple sclerosis(MS) and other demyelinating conditions, and asthma or other pulmonaryconditions in which an immune or inflammatory response is involved (forexample, infection-associated airway hyperactivity, granulomatous lungdisease, emphysema and chronic fibrosing alveolitis and acute hyperoxiclung damage).

IL-1 mediated cellular signaling often involves a molecular activationcascade, during which a receptor propagates a ligand-receptor mediatedsignal by specifically activating intracellular kinases whichphosphorylate target substrates. These substrates can themselves bekinases which become activated following phosphorylation. Alternatively,they can be adaptor molecules that facilitate down stream signalingthrough protein-protein interaction following phosphorylation.Regardless of the nature of the substrate molecule(s), expressedfunctionally active versions of IL-1 eta and its binding partners can beused to identify what substrate(s) were recognized and activated by thepolypeptides of the invention. As such, these novel polypeptides can beused as reagents to identify novel molecules involved in signaltransduction pathways.

Moreover, as described herein, IL-1 eta can be used to identifyantagonists of such signaling pathways. Therefore, administration ofIL-1 eta antagonists will have therapeutic application in blockinginflammatory responses, including the activation of transcriptionfactors NFkappaB and API, the protein kinases Jun N-terminal kinase andp38 MAP kinase, the enzymes COX-2 leading to prostaglandin productionand iNOS leading to nitric oxide production, and inflammation ingeneral. Such signaling pathways have been shown to be involved insepsis, septic, toxic or hemorhagic shock and acute respiratorydistress, such as that which occurs in inhalational anthrax. Antagonistsof IL-1 eta can be used in combination with other agents in thetreatment of inflammatory dysregulation syndromes, including for exampleinhibitors of TNFalpha, inhibitors of other members of the IL-1 family,corticosteroids, and inhibitors of other mediators of inflammation suchas macrophage migration inhibitory factor, and/or inhibitors ofcell-surface receptors such as CD14 and Toll-like receptors.

Similarly, because IL-1 promotes inflammatory responses and is involvedin the causation and maintenance of inflammatory and/or autoimmunediseases, antagonists of IL-1 eta are useful in inhibiting or treatinginflammatory and/or autoimmune disease. Thus, IL-1 eta antagonists willbe useful in treating arthritic conditions that have an inflammatory orautoimmune component, for example, rheumatoid arthritis and/orankylosing spondylitis; inflammatory bowel disease, including Crohn'sDisease and ulcerative colitis, and psoriasis (including psoriaticarthritis). Other inflammatory and/or autoimmune diseases in which IL-1eta is implicated include pulmonary conditions relating to an immune orinflammatory response and/or in which airway hyperreactivity plays arole, for example, asthma, infection-associated airway hyperactivity,granulomatous lung disease, emphysema and chronic fibrosing alveolitisand acute hyperoxic lung damage, and demyelinating conditions that havean inflammatory or autoimmune component, for example, multiple sclerosisand/or chronic inflammatory demyelinating polyneuropathy. Accordingly,antagonists of IL-1 eta will also be useful in ameliorating theseconditions.

Additional conditions for which an autoimmune and/or inflammatorycomponent is a contributory factor (and thus, for which antagonists ofIL-1 eta are useful) include cardiovascular conditions such as stroke,acute myocardial infarction, unstable angina, arterial restenosis andcongestive heart failure. IL-1 eta antagonists are useful in treating orpreventing osteoporosis and/or osteoarthritis, as well asglomerulonephritis, uveitis, and/or Behcet's syndrome. An autoimmune orinflammatory component also plays a role in the cause or maintenance ofsepsis, acute pancreatitis, diabetes (particularly Type II, insulindependent diabetes), endometriosis, and periodontal disease. Similarly,the inflammatory response causes or exacerbates heat stroke andglaucoma, and the cytokines involved in the immune/inflammatory responseplay a supportive role in neoplastic disease (for example, in multiplemyeloma and/or myeloid leukemia), facilitating the growth of neoplasticcells. Accordingly, IL-1 eta antagonists are useful in treating orameliorating these conditions by downregulating the immune and/orinflammatory response that plays a causative role therein.

Moreover, as disclosed in United States Patent Application 20010026801A1, published Oct. 4, 2001, other syndromes and/or conditions are causedor exacerbated by localized production of proinflammatory cytokines.Accordingly, antagonists of IL-1 eta can be administered locally toameliorate a localized inflammatory and/or autoimmune reaction. Suchlocalized reactions occur, for example, in neurological disorders due toa herniated nucleus pulposus (herniated disk), osteoarthritis, otherforms of arthritis, disorders of bone, disease, and/or trauma causingdamage to the optic nerve, other cranial nerves, spinal cord, nerveroots, or peripheral nerves. Moreover, trauma, injury, compression anddisease can affect individual nerves, nerve roots, the spinal cord, orlocalized areas of muscle. Disorders for which localized administrationof antagonists of IL-1 are useful include spinal cord injury, spinalcord compression, spinal stenosis, carpal tunnel syndrome, glaucoma,Bell's palsy, localized muscular disorders (including acute musclepulls, muscle sprains, muscle tears, and muscle spasm), Alzheimer'sdisease and post-herpetic neuralgia. Localized anti-inflammatory agentswill also be useful for treatment of conditions in which fascia,tendons, ligaments or other structures of a joint, and/or otherconnective tissues are injured and/or inflamed (for example, tendonitis,bursitis, strained, sprained or torn ligaments, fascitis, etc.). Usefulantagonists for localized administration in the aforementionedconditions includes localized administration of polypeptide compositionsas well as nucleic acid compositions, as previously described herein.

Antibodies

Antibodies that are immunoreactive with the polypeptides of theinvention are provided herein. Such antibodies specifically bind to thepolypeptides via the antigen-binding sites of the antibody (as opposedto non-specific binding). Thus, the polypeptides, fragments, variants,fusion proteins, etc., as set forth above may be employed as“immunogens” in producing antibodies immunoreactive therewith. Morespecifically, the polypeptides, fragment, variants, fusion proteins,etc. contain antigenic determinants or epitopes that elicit theformation of antibodies.

These antigenic determinants or epitopes can be either linear orconformational (discontinuous). Linear epitopes are composed of a singlesection of amino acids of the polypeptide, while conformational ordiscontinuous epitopes are composed of amino acids sections fromdifferent regions of the polypeptide chain that are brought into closeproximity upon protein folding (C. A. Janeway, Jr. and P. Travers,Immuno Biology 3:9 (Garland Publishing Inc., 2nd ed. 1996)). Becausefolded proteins have complex surfaces, the number of epitopes availableis quite numerous; however, due to the conformation of the protein andsteric hinderances, the number of antibodies that actually bind to theepitopes is less than the number of available epitopes (C. A. Janeway,Jr. and P. Travers, Immuno Biology 2:14 (Garland Publishing Inc., 2nded. 1996)). Epitopes may be identified by any of the methods known inthe art.

Thus, one aspect of the present invention relates to the antigenicepitopes of the polypeptides of the invention. Such epitopes are usefulfor raising antibodies, in particular monoclonal antibodies, asdescribed in more detail below. Additionally, epitopes from thepolypeptides of the invention can be used as research reagents, inassays, and to purify specific binding antibodies from substances suchas polyclonal sera or supernatants from cultured hybridomas. Suchepitopes or variants thereof can be produced using techniques well knownin the art such as solid-phase synthesis, chemical or enzymatic cleavageof a polypeptide, or using recombinant DNA technology.

As to the antibodies that can be elicited by the epitopes of thepolypeptides of the invention, whether the epitopes have been isolatedor remain part of the polypeptides, both polyclonal and monoclonalantibodies may be prepared by conventional techniques. See, for example,Monoclonal Antibodies, Hybridomas: A New Dimension in BiologicalAnalyses, Kennet et al. (eds.), Plenum Press, New York (1980); andAntibodies: A Laboratory Manual, Harlow and Land (eds.), Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1988).

Hybridoma cell lines that produce monoclonal antibodies specific for thepolypeptides of the invention are also contemplated herein. Suchhybridomas may be produced and identified by conventional techniques.One method for producing such a hybridoma cell line comprises immunizingan animal with a polypeptide; harvesting spleen cells from the immunizedanimal; fusing said spleen cells to a myeloma cell line, therebygenerating hybridoma cells; and identifying a hybridoma cell line thatproduces a monoclonal antibody that binds the polypeptide. Themonoclonal antibodies may be recovered by conventional techniques.

The monoclonal antibodies of the present invention include chimericantibodies, e.g., humanized versions of murine monoclonal antibodies.Such humanized antibodies may be prepared by known techniques and offerthe advantage of reduced immunogenicity when the antibodies areadministered to humans. In one embodiment, a humanized monoclonalantibody comprises the variable region of a murine antibody (or just theantigen binding site thereof) and a constant region derived from a humanantibody. Alternatively, a humanized antibody fragment may comprise theantigen binding site of a murine monoclonal antibody and a variableregion fragment (lacking the antigen-binding site) derived from a humanantibody. Procedures for the production of chimeric and furtherengineered monoclonal antibodies include those described in Riechmann etal. (Nature 332:323, 1988), Liu et al. (PNAS 84:3439, 1987), Larrick etal. (Bio/Technology 7:934, 1989), and Winter and Harris (TIPS 14:139,May, 1993). Procedures to generate antibodies transgenically can befound in GB 2,272,440, U.S. Pat. Nos. 5,569,825 and 5,545,806 andrelated patents claiming priority therefrom, all of which areincorporated by reference herein.

Antigen-binding fragments of the antibodies, which may be produced byconventional techniques, are also encompassed by the present invention.Examples of such fragments include, but are not limited to, Fab andF(ab′)₂ fragments. Antibody fragments and derivatives produced bygenetic engineering techniques are also provided.

In one embodiment, the antibodies are specific for the polypeptides ofthe present invention and do not cross-react with other proteins.Screening procedures by which such antibodies may be identified are wellknown, and may involve immunoaffinity chromatography, for example.

Uses Thereof

The antibodies of the invention can be used in assays to detect thepresence of the polypeptides or fragments of the invention, either invitro or in vivo. The antibodies also may be employed in purifyingpolypeptides or fragments of the invention by immunoaffinitychromatography.

Those antibodies that additionally can block binding of the polypeptidesof the invention to the binding partner may be used to inhibit abiological activity that results from such binding. Such blockingantibodies may be identified using any suitable assay procedure, such asby testing antibodies for the ability to inhibit binding of IL-1 eta tocertain cells expressing the IL-1 eta receptors. Alternatively, blockingantibodies may be identified in assays for the ability to inhibit abiological effect that results from polypeptides of the inventionbinding to their binding partners to target cells. Antibodies may beassayed for the ability to inhibit IL-1 eta-mediated, or bindingpartner-mediated cell lysis, for example.

Such an antibody may be employed in an in vitro procedure, oradministered in vivo to inhibit a biological activity mediated by theentity that generated the antibody. Disorders caused or exacerbated(directly or indirectly) by the interaction of the polypeptides of theinvention with the binding partner thus may be treated. A therapeuticmethod involves in vivo administration of a blocking antibody to amammal in an amount effective in inhibiting a binding partner-mediatedbiological activity. Monoclonal antibodies are generally preferred foruse in such therapeutic methods. In one embodiment, an antigen-bindingantibody fragment is employed.

Antibodies may be screened for agonistic (i.e., ligand-mimicking)properties. Such antibodies, upon binding to cell surface receptor,induce biological effects (e.g., transduction of biological signals)similar to the biological effects induced when IL-1 binds to cellsurface IL-1 receptors. Agonistic antibodies may be used to activatevascular endothelial cells and lymphocytes, induce local tissuedestruction and fever (Janeway et al., 1996), stimulate macrophages andvascular endothelial cells to produce IL-6, and upregulate molecules onthe surface of vascular endothelial cells.

Compositions comprising an antibody that is directed againstpolypeptides of the invention, and a physiologically acceptable diluent,excipient, or carrier, are provided herein. Suitable components of suchcompositions are as described above for compositions containingpolypeptides of the invention.

Also provided herein are conjugates comprising a detectable (e.g.,diagnostic) or therapeutic agent, attached to the antibody. Examples ofsuch agents are presented above. The conjugates find use in in vitro orin vivo procedures.

The following examples are offered by way of illustration, and not byway of limitation. Those skilled in the art will recognize thatvariations of the invention embodied in the examples can be made,especially in light of the teachings of the various references citedherein, the disclosures of which are incorporated by reference in theirentirety.

EXAMPLE 1 Isolation of the IL-1 ETA Polynucleotides

Human genomic DNA containing the upstream portion of IL-1 eta cDNA asdefined in EP O879889A2 was cloned and extended in the 3′ direction. Thegenomic DNA was sequenced and examined for potential homology to theC-terminal section of IL-1 family members. A region with the potentialto code with homology to the C-terminal section of IL-1 family memberswas located and is disclosed as polynucleotides 375 to 585 of SEQ. ID.NO.:1. PCR primers were synthesized containing the stop codon in the 3′or reverse primer, and the initiating ATG of the IL-1 eta cDNA (SEQ. ID.NO.:1 of EP 0879889A2) in the 5′ or sense primer. Using these primers,IL-1 eta cDNA was amplified from first stand cDNA made from human tonsilmRNA. PCR was preformed using standard protocols.

EXAMPLE 2 Use of Purified IL-1 eta Polypeptides

Serial dilutions of IL-1 eta-containing samples (in 50 mM NaHCO₃,brought to pH 9 with NaOH) are coated onto Linbro/Fitertek 96 well flatbottom E.I.A. microtitration plates (ICN Biomedicals Inc., Aurora, Ohio)at 100:1/well. After incubation at 4° C. for 16 hours, the wells arewashed six times with 200:1 PBS containing 0.05% Tween-20 (PBS-Tween).The wells are then incubated with FLAG®-binding partner at 1 mg/ml inPBS-Tween with 5% fetal calf serum (FCS) for 90 minutes (100:1 perwell), followed by washing as above. Next, each well is incubated withthe anti-FLAG® (monoclonal antibody M2 at 1 mg/ml in PBS-Tweencontaining 5% FCS for 90 minutes (100:1 per well), followed by washingas above. Subsequently, wells are incubated with a polyclonal goatanti-mIgG1-specific horseradish peroxidase-conjugated antibody (a 1:5000dilution of the commercial stock in PBS-Tween containing 5% FCS) for 90minutes (100:1 per well). The HRP-conjugated antibody is obtained fromSouthern Biotechnology Associates, Inc., Birmingham, Ala. Wells then arewashed six times, as above.

For development of the ELISA, a substrate mix [100:1 per well of a 1:1premix of the TMB Peroxidase Substrate and Peroxidase Solution B(Kirkegaard Perry Laboratories, Gaithersburg, Md.)] is added to thewells. After sufficient color reaction, the enzymatic reaction isterminated by addition of 2 N H₂SO₄ (50:1 per well). Color intensity(indicating ligand receptor binding) is determined by measuringextinction at 450 nm on a V Max plate reader (Molecular Devices,Sunnyvale, Calif.).

EXAMPLE 3 Amino Acid Sequence

The amino acid sequence of IL-1 eta was determined by translation of thenucleotide sequence of SEQ ID NO:1. The coding region includesnucleotide residues 112–585.

EXAMPLE 4 DNA and Amino Acid Sequences

The nucleotide sequence of the isolated IL-1 eta and the amino acidsequence encoded thereby, are presented in SEQ ID NOs:1 and 2. Thesequence of the IL-1 eta DNA fragment isolated by PCR corresponds tonucleotides 1 to 585 of SEQ ID NO:1. Nucleotide residues 112–585 encodeamino acids 1 to 157 of SEQ ID NO:2.

The amino acid sequence of SEQ ID NO:2 bears significant homology toother known IL-1 ligand family members.

EXAMPLE 5 Monoclonal Antibodies that Bind Polypeptides of the Invention

This example illustrates a method for preparing monoclonal antibodiesthat bind IL-1 eta. Suitable immunogens that may be employed ingenerating such antibodies include, but are not limited to, purifiedIL-1 eta polypeptide or an immunogenic fragment thereof such as theextracellular domain, or fusion proteins containing IL-1 eta (e.g., asoluble IL-1 eta/Fc fusion protein).

Purified IL-1 eta can be used to generate monoclonal antibodiesimmunoreactive therewith, using conventional techniques such as thosedescribed in U.S. Pat. No. 4,411,993. Briefly, mice are immunized withIL-1 eta immunogen emulsified in complete Freund's adjuvant, andinjected in amounts ranging from 10–100 g subcutaneously orintraperitoneally. Ten to twelve days later, the immunized animals areboosted with additional IL-1 eta emulsified in incomplete Freund'sadjuvant. Mice are periodically boosted thereafter on a weekly tobi-weekly immunization schedule. Serum samples are periodically taken byretro-orbital bleeding or tail-tip excision to test for IL-1 etaantibodies by dot blot assay, ELISA (Enzyme-Linked Immunosorbent Assay)or inhibition of IL-1 eta receptor binding.

Following detection of an appropriate antibody titer, positive animalsare provided one last intravenous injection of IL-1 eta in saline. Threeto four days later, the animals are sacrificed, spleen cells harvested,and spleen cells are fused to a murine myeloma cell line, e.g., NS1 orpreferably P3x63Ag8.653 (ATCC CRL 1580). Fusions generate hybridomacells, which are plated in multiple microtiter plates in a HAT(hypoxanthine, aminopterin and thymidine) selective medium to inhibitproliferation of non-fused cells, myeloma hybrids, and spleen cellhybrids.

The hybridoma cells are screened by ELISA for reactivity againstpurified IL-1 eta by adaptations of the techniques disclosed in Engvallet al., (Immunochem. 8:871, 1971) and in U.S. Pat. No. 4,703,004. Apreferred screening technique is the antibody capture techniquedescribed in Beckmann et al., (J. Immunol. 144:4212, 1990). Positivehybridoma cells can be injected intraperitoneally into syngeneic BALB/cmice to produce ascites containing high concentrations of anti-IL-1 etamonoclonal antibodies. Alternatively, hybridoma cells can be grown invitro in flasks or roller bottles by various techniques. Monoclonalantibodies produced in mouse ascites can be purified by ammonium sulfateprecipitation, followed by gel exclusion chromatography. Alternatively,affinity chromatography based upon binding of antibody to Protein A orProtein G can also be used, as can affinity chromatography based uponbinding to IL-1 eta.

EXAMPLE 6 Northern Blot Analysis

The tissue distribution of IL-1 eta is investigated by Northern blotanalysis, as follows. An aliquot of a radiolabeled riboprobe is added totwo different human multiple tissue Northern blots (Clontech, Palo Alto,Calif.; Biochain, Palo Alto, Calif.). The blots are hybridized in 10×Denhardts, 50 mM Tris pH 7.5, 900 mM NaCl, 0.1% Na pyrophosphate, 1%SDS, 200 μg/mL salmon sperm DNA. Hybridization is conducted overnight at63° C. in 50% formamide as previously described (March et al., Nature315:641–647, 1985). The blots are then washed with 2×SSC, 0.1% SDS at68° C. for 30 minutes. The cells and tissues with the highest levels ofIL-1 eta mRNA are determined by comparison to control probing with aβ-actin-specific probe.

Expression of IL-eta was also analyzed in several animal models of humandisease by conventional real-time polymerase chain reaction (RT-PCR)substantially as described in U.S. Ser. No. 09/876,790, filed Jun. 6,2001, and/or by TaqMan® RT-PCR (Applied Biosystems, Foster City,Calif.).). Total RNA from small or large intestine (colitis models:DSS-induced colitis, anti-CD-3 induced ileitis and MdrKO spontaneouscolitis), spinal cord (multiple sclerosis [MS] models: EAE using SJLmice injected with PLP), or lung (asthma model: BALB/c/OVA-inducedasthma model) was used to make first strand cDNA. The level ofexpression was subjectively scored as a function of relative ethidiumbromide staining intensity.

Results of these experiments indicated that expression of IL-1 eta wasupregulated in DSS-induced colitis. Accordingly, IL-1 eta is implicatedin the cause or prolongation of inflammatory bowel disease, andantagonists thereof will be useful in treating or amelioratinginflammatory bowel disease in individuals afflicted with suchconditions. Additionally, IL-1 eta appeared to be upregulated in theearly stages of EAE, indicating that an antagonist thereof may be usefulin treating or ameliorating MS and other demyelinating conditions. IL-1eta was also upregulated in the OVA-induced asthma model, indicatingthat an antagonist thereof may be useful in treating or amelioratingasthma and other pulmonary conditions relating to an immune orinflammatory response.

EXAMPLE 7 Binding Assay

Full length IL-1 eta can be expressed and tested for the ability to bindIL-1 eta receptors. The binding assay can be conducted as follows.

A fusion protein comprising a leucine zipper peptide fused to theN-terminus of a soluble IL-1 eta polypeptide (LZ-IL-1 eta) is employedin the assay. An expression construct is prepared, essentially asdescribed for preparation of the FLAG® (IL-1 eta) expression constructin Wiley et al. (Immunity, 3:673–682, 1995; hereby incorporated byreference), except that DNA encoding the FLAG® peptide was replaced witha sequence encoding a modified leucine zipper that allows fortrimerization. The construct, in expression vector pDC409, encodes aleader sequence derived from human cytomegalovirus, followed by theleucine zipper moiety fused to the N-terminus of a soluble IL-1 etapolypeptide. The LZ-IL-1 eta is expressed in CHO cells, and purifiedfrom the culture supernatant.

The expression vector designated pDC409 is a mammalian expression vectorderived from the pDC406 vector described in McMahan et al. (EMBO J.10:2821–2832, 1991; hereby incorporated by reference). Features added topDC409 (compared to pDC406) include additional unique restriction sitesin the multiple cloning site (mcs); three stop codons (one in eachreading frame) positioned downstream of the mcs; and a T7 polymerasepromoter, downstream of the mcs, that facilitates sequencing of DNAinserted into the mcs.

For expression of full length human IL-1 eta protein, the entire codingregion (i.e., the DNA sequence presented in SEQ ID NO:1) is amplified bypolymerase chain reaction (PCR). The template employed in the PCR is thecDNA clone isolated from tonsil first strand cDNA, as described inexample 1. The isolated and amplified DNA is inserted into theexpression vector pDC409, to yield a construct designated pDC409-IL-1eta.

LZ-IL-1 eta polypeptide is employed to test the ability to bind to hostcells expressing recombinant or endogenous IL-1 eta receptors, asdiscussed above. Cells expressing IL-1 eta receptor are cultured in DMEMsupplemented with 10% fetal bovine serum, penicillin, streptomycin, andglutamine. Cells are incubated with LZ-IL-1 eta (5 mg/ml) for about 1hour. Following incubation, the cells are washed to remove unboundLZ-IL-1 eta and incubated with a biotinylated anti-LZ monoclonalantibody (5 mg/ml), and phycoerythrin-conjugated streptavidin (1:400),before analysis by fluorescence-activated cell scanning (FACS). Thecytometric analysis was conducted on a FACscan (Beckton Dickinson, SanJose, Calif.).

The cells expressing IL-1 eta receptors showed significantly enhancedbinding of LZ-IL-1 eta, compared to the control cells not expressingIL-1 eta receptors.

EXAMPLE 8 Expression Analysis

First strand cDNAs present in Clontech (Palo Alto, Calif.) HumanMultiple Tissue cDNA Panels I (Cat. # K1420-1) and II (Cat. #K1421-1)and the Human Immune Panel (Cat. #K1426-1) were screened by PCRamplification using primers (sense: ACATCATGAACCCACAACGGGAGGCAGCAC (SEQID NO:6); antisense: CTCTATCCTGGAACCAGCCACCCACAGC (SEQ ID NO:7)). Theprimers were designed to span introns so that products arising fromgenomic DNA and cDNA could be distinguished. In some cases, nestedprimers (sense: CCAAATCCTATGCTATTCGTGATTCTCGAC (SEQ ID NO:8); antisense:GGMTTTATTCCACAGAATCTAAGTAGAAG (SEQ ID NO:9)) were used in a second PCRreaction. The presence of an amplification product for each gene/tissuecombination was determined by analysis on agarose gels stained withethidium bromide.

Alternatively, individual cell types from human peripheral blood wereisolated and stimulations were performed (Kubin et al., Blood83(7):1847–55 (1994); Kubin et al., J Exp Med 180(1):211–22 (1994)). NKcells were incubated with IL-12 (R&D Biosystems; 1 ng/ml) for either 2hours or 4 hours. T cells were unstimulated or stimulated with anti-CD3(OKT-3 antibody, immobilized on plastic at 5 ng/ml) or with thecombination of anti-CD3 and anti-CD28 (the anti-CD28 antibody was CD248used in soluble form as a 1:500 dilution of ascites fluid), for 30minutes or 4 hours. Monocytes were unstimulated, or stimulated with LPS(Sigma; 1 ug/ml) for 2 or 3 hours. B cells were unstimulated, orstimulated with the combination of 0.05% SAC and 500 ng/ml CD40L trimer(Immunex) and 5 ng/ml IL-4 (Immunex) for 3.5 or 4 hours. Dendritic cellswere stimulated with LPS as for monocytes, for 2 or 4 hours. Afterisolation of RNA and synthesis of first strand cDNA, PCR amplificationsand gel analysis were performed.

Table I demonstrates the expression of IL-1 eta in lymphoid organs. A“-” indicates that the mRNA was looked for but not found. Positiveresults derived by PCR analysis for a panel of first strand cDNAs(Clontech) are designated by an “A”.

EXAMPLE 9 Binding Assay

This example describes a type of binding assay utilizing the inventiveproteins. A recombinant expression vector containing the binding partnercDNA is constructed using methods well known in the art. CV1-EBNA-1cells in 10 cm² dishes are transfected with the recombinant expressionvector. CV-1/EBNA-1 cells (ATCC CRL 10478) constitutively express EBVnuclear antigen-1 driven from the CMV immediate-early enhancer/promoter.CV1-EBNA-1 was derived from the African Green Monkey kidney cell lineCV-1 (ATCC CCL 70), as described by McMahan et al. (EMBO J. 10:2821,1991).

The transfected cells are cultured for 24 hours, and the cells in eachdish then are split into a 24-well plate. After culturing an additional48 hours, the transfected cells (about 4×10⁴ cells/well) are washed withBM-NFDM, which is binding medium (RPMI 1640 containing 25 mg/ml bovineserum albumin, 2 mg/ml sodium azide, 20 mM Hepes pH 7.2) to which 50mg/ml nonfat dry milk has been added. The cells then are incubated for 1hour at 37° C. with various concentrations of, for example, a solublepolypeptide/Fc fusion protein made as set forth above. Cells then arewashed and incubated with a constant saturating concentration of a¹²⁵I-mouse anti-human IgG in binding medium, with gentle agitation for 1hour at 37° C. After extensive washing, cells are released viatrypsinization.

The mouse anti-human IgG employed above is directed against the Fcregion of human IgG and can be obtained from Jackson ImmunoresearchLaboratories, Inc., West Grove, Pa. The antibody is radioiodinated usingthe standard chloramine-T method. The antibody will bind to the Fcportion of any polypeptide/Fc protein that has bound to the cells. Inall assays, non-specific binding of ¹²⁵I-antibody is assayed in theabsence of the Fc fusion protein/Fc, as well as in the presence of theFc fusion protein and a 200-fold molar excess of unlabeled mouseanti-human IgG antibody.

Cell-bound ¹²⁵I-antibody is quantified on a Packard Autogamma counter.Affinity calculations (Scatchard, Ann. N.Y. Acad. Sci. 51:660, 1949) aregenerated on RS/1 (BBN Software, Boston, Mass.) run on a Microvaxcomputer.

EXAMPLE 10 Activation of Signaling Molecules in Human Cells

The following describes tests and results that are carried out evaluatethe induction of some of the same signaling molecules involved in stressresponses as are activated by IL-1 alpha, IL-1 beta and otherinflammatory cytokines.

Human IL-1 eta is transfected into COS-1 cells. Several days after thetransfection, conditioned medium (containing the transiently expressedIL-1 eta) is harvested. Test cells are incubated with this conditionedmedium, or alternatively with conditioned medium from COS-1 cellstransfected with the empty expression vector. Approximately 10 minutesfollowing the incubation, cell extracts are prepared from the testcells, and the presence of activated signaling molecules is assayed bythe use of antibodies specific for the phosphorylated forms of IKBalpha(phosphorylation on Ser32), p38 MAP kinase (phosphorylation on Thr180and Tyr182), and Stress-Activated Protein Kinase (SAPK/JNK)(phosphorylation on Thr183/Tyr185). The antibodies may be obtained fromcommercial sources, such as New England Biolabs, Beverly, Mass. Thesesignal transduction molecules are known to be involved in a wide rangeof cellular responses to stimuli such as UV irradiation, endotoxin, andinflammatory cytokines including IL-1 beta. phosphorylation of one ormore of these molecules indicates that IL-1 eta is involved in stressresponse signaling pathways.

EXAMPLE 11 Activation of Cell Surface Molecules in Human Cells

The following describes tests that are carried out to evaluate theability of IL-1 eta to induce cell surface molecules involved in stressresponses (such as those that are induced by IL-1 alpha, IL-1 beta andother inflammatory cytokines).

Human IL-1 eta is transfected into COS-1 cells. Several days after thetransfection, conditioned medium (containing the transiently expressedIL-1 eta) is harvested. Human foreskin fibroblast (HFF) cells areincubated for 18 hours at 37 degrees C. with this conditioned mediumdiluted 1:1 with fresh 0.5% serum-containing medium, or alternativelywith conditioned medium from control COS-1 cells transfected with theempty expression vector, diluted 1:1 with fresh 0.5% serum-containingmedium.

Following treatment with the conditioned medium from COS-1 cells, theHFF cells are washed twice with PBS and removed from the tissue culturevessel with versene (non-trypsin reagent). Cell-surface ICAM-1 levelsare measured by staining with anti-CD54-PE antibody (Pharmingen, SanDiego, Calif.) on ice for one hour followed by washing and FACS-baseddetection. An increase in the level of cell-surface ICAM-1 indicatesthat IL-1 eta is involved in upregulating cell-surface molecules thatare induced during stress response.

EXAMPLE 12 Modulation of Cytokine Levels by IL-1 eta

The following describes tests that are carried out to evaluate inductionof cytokine secretion in dendritic cells or other cells capable ofsecreting cytokines.

Monocyte-derived dendritic cells (MoDC) are obtained essentially asdescribed by Pickl et al. (J. Immunol. 157:3850, 1996). Briefly, highlypurified CD14(bright) peripheral blood monocytic cells are obtained fromperipheral blood using an AutoMACS cell sorting system and anti-CD 14magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Themonocytic cells are cultured in the presence of IL-4 and GM-CSF forseven days to yield MoDC. Similar techniques are used to obtainedpurified or enriched populations of other cytokine-secreting cells, forexample lymphocytes or granulocytes

Cells are treated for two to three days in the presence or absence ofIL-1 eta at varying concentrations; lipopolysaccharide (LPS) at 10 ng/mlis used as a positive control; heat-inactivated IL-1 eta (heated at 100degrees C. for 30 minutes) may be used as a negative control. Cells areseparated from the supernatant medium by centrifugation.

The supernatant medium is analyzed for soluble cytokine levels using asuitable assay (for example, the Luminex® multi-plex cytokine assay;Luminex Corporation, Austin, Tex.). Following culture, the supernatantis harvested and assayed for several cytokines including IL-10, IL-2,IL-4, IL-6, IL-8, IL-12 (p70 heterodimer), TNF-alpha, IFN-gamma, andGM-CSF.

For analysis of the induction of cytokine mRNA, the cells are harvestedand total RNA is isolated (for example, using an RNeasy® Total RNASystem mini-kit, QIAGEN, Venlo, The Netherlands) and analyzed in asuitable, real-time quantitative polymerase chain reaction (PCR)analysis. Quantitative RT-PCR is performed using the ABI PRISM® 7700Sequence Detection System (Applied Biosystems, Foster City, Calif.) andTaqMan® reagents (Applied Biosystems). An increase in the levels of oneor more cytokines and/or induction of one or more cytokine mRNAsindicates that IL-1 eta upregulates cytokines that are involved in theinflammatory and/or immune response.

EXAMPLE 13 Effect of IL-1 eta on Mixed Lvmphocyte Reaction (MLR)

The following describes tests carried out to evaluate the effects ofIL-1 eta on TNF-alpha, IFN-gamma, and IL-10 secretion in a mixedleukocyte reaction (MLR) assay.

Briefly, highly purified CD14(bright) peripheral blood monocytic cellsare obtained from peripheral blood using an AutoMACS cell sorting systemand anti-CD14 magnetic microbeads (Miltenyi Biotec, Bergisch Gladbach,Germany). The monocytic cells are cultured in the presence of IL-4 andGM-CSF for seven days to yield MoDC. Purified CD3+ allogeneic T cellsare obtained from freshly drawn blood using an AutoMACS cell sorting andanti-CD3 magnetic microbeads system (Miltenyi Biotec).

The allogeneic T cells are then mixed with MoDCs at a 1:10 MoDC:T ratioin quadruplicate in the presence of IL-1 eta at varying concentrationsfrom 5 ng/ml to 200 ng/ml, or control preparations. The ensuing mixedlymphocyte reaction (MLR) is allowed to proceed for four days, andsupernatants are harvested and assayed for TNF-alpha, IFN-gamma, andIL-10 using a suitable assay as described previously (for example, theLuminex® multi-plex cytokine assay, DELFIA® or ELISA substantially asdescribed below).

EXAMPLE 14 Cytokine ELISA

The following describes an Enzyme-Linked Immunosorbent Assay (ELISA)that is useful to detect and/or quantitate secreted proteins. TheExample describes an assay specific for IL-10; those of skill in the artwill recognize that a similar assay could be used to detect othermolecules.

ELISA plates (for example, Costar® EIA/RIA 96 well easy wash plates,Corning Incorporated Life Sciences, Acton, Mass.) are coated overnightwith 100 microliter of a 2 micrograms/ml mixture of Rat-anti-huIL-10capture antibody (BD Pharmingen, San Diego, Calif.) in binding solution(0.1M NaH₂PO₄, pH 9.0) at 4 degrees C. Plates are washed with washbuffer (phosphate buffered saline, or PBS, 0.5% Tween 20) four times(400 microliters/well/wash), then one time with PBS without Tween.Plates were blocked with 100 microliters of 5% non-fat dry milk in PBSfor 1 hour at room temperature (RT), and then washed with wash buffersix times.

Samples and controls are added to separate wells (100 microliters/well);serial dilutions of a standard protein, recombinant HuIL-10 (BDPharmingen) in PBS+3% BSA (starting at 10 ng/ml in 3-fold dilutionsthrough 7 points as a standard curve, with an eighth point as a blank)is used to generate a standard curve for quantitation. The plates areincubated for one hour at RT, then washed with wash buffer six times aspreviously described, and incubated with biotinylated-rat-anti-HuIL-10(BD Pharmingen; 100 microliters/well of a 200 ng/ml mixture in PBS+3%BSA) for one hour at RT. The plates are then washed six times with washbuffer as before, and streptavidin-conjugated horse radish peroxidase(SA-HRP; Zymed Laboratories, Inc., South San Francisco, Calif.; 100microliters/well of a 1:4000 dilution in PBS+3% BSA) is added.

After incubating at RT for 30 minutes, the plates are washed for thefinal time as described above, and color is developed by adding 100microliters/well of Tetramethylbenzidene (TMB) substrate (a 1:1 mixtureof TMB Peroxidase Substrate: Peroxidase Solution, Kirkegaard & PerryLaboratories, Inc., Gaithersburg, Md.). The plates are incubated for 30minutes at RT, at which time color development is stopped with 100microliters/well of 2N H₂SO₄. The plates are read at 450 nm wavelengthon a Molecular Dynamics (Molecular Dynamics, Sunnyvale, Calif.) platereader, a standard curve is prepared, and the quantity of IL-10 in thesamples determined by comparison to the standard curve.

EXAMPLE 15 Cytokine DELFIA

The following describes a DELFIA® (dissociated enhanced lanthanidefluoroimmunoassay; PerkinElmer LifeSciences, Wallac Oy., Turku, Finland)that is useful to detect and/or quantitate secreted proteins. TheExample describes an assay specific for IL-10; those of skill in the artwill recognize that a similar assay could be used to detect othermolecules.

Briefly, DELFIA® plates (i.e., Costar® high binding 96-well plates,Corning Incorporated Life Sciences, Acton, Mass.) are coated with adetection (or capture) antibody (preferably a monoclonal antibody; 50microliters of antibody solution containing 2 micrograms antibody/ml inPBS) at 4 degrees C. for 24 hours. Plates are washed with wash buffer(phosphate buffered saline, or PBS, 0.05% Tween 20) four times (300microliters/well/wash), then used in an assay or stored.

Fifty microliters each of test supernatants and cell specific controlsare added to separate wells of an antibody-coated plate; dilutions ofstandard proteins are used to generate a standard curve forquantitation. Test supernatants and controls are incubated in theantibody coated plate to allow binding of cytokine to the antibody.Plates are then washed and a polyclonal biotinylated detection antibodyis added at a concentration of 10 nM in 50 microliters and incubated toallow binding to the captured cytokine. Plates are washed andStreptavidin-Europium (Eu) is added to the plate at a finalconcentration of 1 nM (0.06 micrograms/ml) in 50 microliters andincubated to allow binding to the biotinylated detection antibody.Plates are again washed and 100 microliters of enhancement solution isadded to bind the Eu. The Eu in solution is then detected by timeresolved fluorescence and the amount of cytokine secreted can bequantitated relative to standards which are added to each plate.

DELFIA® is amenable to full or partial automation (for example, using aSagian Bioassay core system, Beckman Coulter, Inc., Fullerton, Calif.,in combination with a plate reader such as a VICTOR2™, PerkinElmerLifeSciences), thereby rendering it useful for high-throughput testing.

EXAMPLE 16 Mouse Inflammatory Bowel Disease Models

This example describes several mouse models of inflammatory boweldisease (IBD), which includes Crohn's Disease and ulcerative colitis.Inflammatory bowel disease in animals can either occur spontaneously orcan be experimentally induced. It is necessary to exercise care whenselecting IBD models to study to ensure that the particular modelselected appropriately represents the relevant stage of the inflammatoryprocess under investigation. Particularly useful models of IBD include:

A. Oral Administration of Dextran Sulfate Sodium (DSS)

The DSS induction model can be used to induce either chronic or acuteIBD. In the acute protocol, mice are given DSS (preferably with amolecular weight of 40 Kd; from 2% to 8%) in their drinking water forfrom one to eight days. The percent DSS and the duration of inductionwill vary depending on the strain of mouse used (for example, C3H/HeJ,C3H/He/Bir, NOD and NOD/SCID mice are highly susceptible, DBA/2,C57BL/6. BALB/c and 129/SvJ mice are moderately susceptible, withvarying degrees of susceptibility relative to each other, FVB mice aremoderately resistant, and NON/Ltj mice are resistant to DSS inducedcolitis). In the acute model, DSS is withdrawn after the inductionphase. To induce chronic colitis, 2–8% DSS is administered for from 5 toseven days followed by administration of water for ten days; this cycleis repeated three to four times.

DSS-induced colitis is marked by profound inflammation in the colon ofanimals characterized by crypt destruction, mucosal ulceration, erosionsand infiltration of lymphocytes and neutrophils into the mucosal tissue.Histopathologic changes are individually scored as 0 (no findings), 1(minimal), 2 (mild), 3 (moderate), 4 (severe) for each of the followingparameters: increased lymphocytes, increased neutrophils, ulceration,edema, crypt degeneration, and crypt regeneration. Total lesion score,crypt length and number of ulcers are also determined and used to gageseverity of colitis.

B. Anti-CD3-Induced Ileitis

Mice (for example, BALB/c, C57BL/6 or MPJ mice, 6–16 weeks of age) aregiven a single intraperitoneal (i.p.) injection of anti-CD3epsilonantibody or control Ab (50 micrograms diluted in 500 microliters PBS, pH7.4). In wildtype mice such as those listed above, this treatmentreliably induces diarrhea without being lethal. Immunosuppressants suchas cyclosporin A (CsA, 50 mg/kg) or dexamethasone (Dex, 50 mg/kg) may begiven i.p. either as a single dose at the same time as anti-CD3antibody, or daily for a total of three injections beginning at the timeof anti-CD3 injection, as control molecules that downregulate anyensuing immune response and prevent or ameliorate anti-CD3-inducedileitis.

Mice are monitored for clinical signs of ileitis; mice may be sacrificedat varying time points for histopathologic analysis and/or testing byother means to evaluate apoptosis in gut tissue. For histopathology,hematoxylin and eosin (H&E) stained tissue sections of paraffin embeddedintestinal specimens are graded in a blinded fashion, for example byusing a quantitative histology score based on the frequency of apoptoticepithelial cells within the epithelium and the ratio of villus height tocrypt length. Histological alterations of the small intestinal mucosathat may be observed include a reduced villus height, increasedthickness of the crypt region, loss of Paneth cells, goblet cells andIEL in the epithelial layer and severe morphologic changes of theepithelial cells. In the villi, the enterocytes may have lost theircolumnar and polarized morphology and become flattened. In the cryptregion, numerous apoptotic bodies may identified in the epithelium.

C. MdrKO Spontaneous Colitis

The MDR gene family was identified by an ability to confer multiple drugresistance in cell lines. Three genes have been identified in rodents(mdr1, mdr2 and mdr3), and two in humans (MDR1, MDR3). The mouse mdr1agene encodes a 170 kDa transmembrane protein that is expressed in manytissues, including intestinal epithelial cells and subsets of lymphoidand hematopoietic cells. Its function in these cells is currentlyunknown, however, mice deficient in mdr1a spontaneously develop colitis.In humans, MDR1 may be associated with IBD susceptibility (Satsangi etal., Nat. Genet. 14:199, 1996; Brant et al., Gastroenterology, 118:A331,2000), while decreased MDR1 expression has been reported in mucosaltissue from both CD and UC patients (Lawrance et al., Hum. Mol. Genet.10: 445, 2001; Farrell et al., Gastroenterology, 118:279, 2000). Mdr1aknockout mice (MdrKO) provide a model of both acute (spontaneous) andchronic (DSS-induced) IBD, similar to that seen in humans, where IBD isgenerally a mixture of both chronic and acute inflammation. Acutecolitis in MdrKO mice is marked by the spontaneous appearance ofdiarrhea and bloody stools in a subset of the mice; chronic colitis isinduced by administering 3% w/v DSS for seven days in drinking water,followed by normal water.

Histopathologic changes are individually scored as 0 (no findings), 1(minimal), 2 (mild), 3 (moderate), 4 (severe) for each of the followingparameters: increased mononuclear cells, increased neutrophils,ulceration, edema, crypt degeneration, and hyperplasia.

D. Helicobacter-Induced Colitis

Various strains of mice with immunologic defects (i.e., IL-10^(−/−)mice, recombinase-activating gene (Rag)1^(−/−) mice, T-cell receptoralpha (TCRalpha)^(−/−) mice) are susceptible to colitis induced byinfection with Helicobacter spp., as described in Burich et al. (Am JPhysiol Gastrointest Liver Physiol 281:G764, 2001). Moreover, luminalbacteria appear to be an important factor contributing to thedevelopment of IBD in mice and humans. Accordingly, introduction ofHelicobacter spp. into immunodeficient mice also serves as an animalmodel of IBD humans (Burich et al. supra). In MdrKO mice, differentspecies of Helicobacter may have different effects on spontaneouscolitis; H. bilis infection induces IBD at a much earlier age, and thephenotypic appearance of Helicobacter-induced disease is similar, butnot identical, to spontaneous IBD. In contrast, there is minimal diseasein H. hepaticus-infected mdrla−/− mice, and H. hepaticus appears todelay onset of spontaneous IBD. Accordingly, those of skill in the artcan utilize a Helicobacter-based model of IBD substantially as describedby Burich et al. supra.

EXAMPLE 17 Mouse Asthma Models

This example describes a mouse model of asthma. Mice (for example,BALB/c) are sensitized with antigen (for example, ovalbumin [OVA]) byintraperitoneal injection of the antigen in alum. Several sensitizationschemes are known in the art; a preferred scheme is to inject 10micrograms of OVA three times at one week intervals (i.e., on day −21,day —14 and day —7). The mice are then challenged with antigen either byaerosol exposure (5% OVA) or intranasal administration (0.1 mg OVA). Thechallenge schedule may be selected from among shorter terms (i.e., dailychallenge on days 1, 2 and 3) or longer terms (i.e., weekly challengefor two to three weeks). The endpoints that are measured can includeairway hyperreactivity, bronchoalveolar lavage (BAL) cell number andcomposition, in vitro draining lung lymph node cytokine levels, serumIgE levels, and histopathologic evaluation of lung tissue. Other animalmodels of asthma are known, and include the use of other animals (forexample, C57BL/6 mice), sensitization schemes (for example, intranasalinoculation, use of other adjuvants or no adjuvants, etc.) and/orantigens (including peptides such as those derived from OVA or otherproteinaceous antigens, ragweed extracts or other extracts such as thoseused in desensitization regimens, etc.).

EXAMPLE 18 Mouse Collagen Induced Arthritis Model

This example describes two mouse models of rheumatoid arthritis, both ofwhich are induced by immunization with collagen (eg., collagen-inducedarthritis or CIA). One model is dependant on tumor necrosis factor(TNF), the other is TNF-independent. Those of skill in the art recognizethat other animals models of rheumatoid arthritis exist, and furtherthat various parameters within the models can be adjusted (see, forexample, Luross and Williams, Immunology 103:407, 2001; Schaller et al.,Nat Immunol 2:74, 2001; Bober et al., Arthritis Rheum 43:2660, 2000; orWeyand, C. M. in Rheumatology (Oxford) 2000 June, pgs: 3–8)).

TNF-dependent CIA is induced in male, wild-type (wt) DBA/1 micesubstantially as a modification of the protocol described by Courtenay,J. S. et al. (Nature 283:666, 1980) by immunization of mice with Type IIcollagen (CII; 100–200 micrograms) in complete Freund's adjuvant (CFA),followed by a booster of CII (200 micrograms) in incomplete Freund'sadjuvant (IFA) approximately three weeks later. In untreated mice, CIAmanifests in the paws, with increasing severity over time.

TNF-independent CIA is induced in male TNF Receptor double knockout(TNFR DKO) mice substantially as described above. TNFR DKO mice are micethat lack functional TNF receptors (both p55 and p75), and are describedin Peschon, et al. (J. Immunol. 160:943, 1998). Briefly, mice lackingfunctional p55 and p75 genes were generated in C57BL/6 background bygene targeting in embryonic stem cells. The TNFR DKO C57BL/6 mice wereback-crossed on to the DBA/1 genetic background to yield mice that werehomozygous for H-2q and were susceptible to development of CIA.

The severity of disease is judged by swelling and joint function of eachpaw, using a score from 0 to 4 (0=normal, no swelling; 1=swelling in 1to 3 digits; 2=mild swelling in ankles, forepaws or more than threedigits; 3=moderate swelling in multiple joints; 4=severe swelling withloss of function). The score for each paw is totaled for a cumulativescore for each mouse; cumulative scores are totaled for the mice in eachexperimental group to yield a mean clinical score.

EXAMPLE 19 Mouse Experimental Allergic Encephalomyelitis Model

This example describes two mouse models of demyelinating conditions;experimental autoimmune encephalomyelitis (or EAE) is designed toduplicate the secondary, immune mediated demyelination that occurs inmultiple sclerosis.

A. Myelin Oligodendrocyte Glycoprotein (MOG)-Induced EAE in C57BL/6 Mice

EAE is induced in female C57BL/6 mice substantially as described byMendel et al. (Eur. J. Immunol. 25:1951–59, 1995) by immunization ofmice with an antigen derived from rat myelin oligodendrocyteglycoprotein (preferably the MOG35-55 peptide described by Mendel etal., supra). Other encephalitogenic antigens may be used, including, forexample, whole spinal chord homogenate, purified whole myelin, myelinbasic protein, proteolipid protein, myelin associated glycoprotein,myelin-associated oligodendrocyte basic protein, or encephalitogenicpeptides derived from these antigens. The disease induction protocol ofMendel et al. may be modified to include the use of a lower dose ofMOG35-55 for immunization (see below), no booster immunization, and theuse of RIBI® adjuvant (Corixa Corporation, Seattle Wash.) instead ofcomplete Freund's adjuvant.

To induce EAE, groups of age and weight-matched mice are given a dose of100 micrograms of rat MOG35-55 emulsified in 0.2 ml RIBI® adjuvant andinjected subcutaneously (for example, at three sites distributed overthe shaved flank of a mouse). To induce EAE with accelerated onset, micemay be given an intravenous injection 500 ng pertussis toxin (ListBiological Laboratory Inc, Campbell, Calif.), administered 48 hoursafter administration of MOG35-55.

B. Proteolipid Protein (PLP)-Induced EAE in SJL Mice

The PLP/SJL model results in a relapsing-remitting course of diseasethat mimics the course often seen in MS; however, SJL mice aresusceptible to anaphylaxis, and care must be given in choosing andadministering therapeutic agents to avoid induction of an anaphylacticresponse. EAE is induced in female SJL mice substantially as describedby McRae et al. et al. (J. Neuroimmunol. 38:229, 1992) by immunizationof mice with an antigen derived from rat proteolipid protein (preferablythe PLP13-151(S) peptide described by McRae et al., supra). Otherencephalitogenic antigens may be used, including, for example, wholespinal chord homogenate, purified whole myelin, myelin basic protein,proteolipid protein, myelin associated glycoprotein myelin-associatedoligodendrocyte basic protein, or encephalitogenic peptides derived fromthese antigens. The disease induction protocol of McRae et al. may bemodified as described above. EAE is reliably induced in SJL/J miceactively immunized with PLP13-151(S) or another, suitable PLP-relatedantigen. Alternatively, EAE can be induced by adoptive transfer ofPLP-specific T cells.

Administration of FIL1 antagonist(s) or control for either or bothmodels is initiated on the day after administration of theencephalitogenic peptide (day 1) and continued through day 11. Varyinginjection schedules can be used to evaluate the efficacy of the FIL1antagonist(s). Each mouse is injected intraperitoneally every other day(or according to the selected injection schedule) with 0.2 mlpyrogen-free phosphate-buffered saline (PBS) or 0.2 ml PBS containingFIL1 antagonist(s) or control. Endotoxin levels are monitored and mustbe less that <10 EU/mg of protein for all reagents. Mice are monitoreddaily for 30 to 35 days for weight loss, disease onset and severity ofclinical signs of EAE by an independent observer blinded to thetreatment groups.

The severity of EAE is assessed using either a standard EAE index systemin which “0” is used to indicate an asymptomatic mouse and clinicalscores ranging from 0.5 to 4 are used to indicate varying degrees ofascending paralysis, or a slightly modified version of the commonly usedEAE scoring system. In the latter system, “0” indicates a mouse with noevidence of disease and scores of 1–5 indicate varying degrees ofascending paralysis as follows: 1, tail paralysis; 2, hind limbweakness; 3, partial hind limb paralysis; 4, complete hind limbparalysis; 5, moribund or dead. The disease protocol described aboveinduces an acute episode of disease in control mice (peak score of 2–4)from which most recover at least partially. Thus the acute episode ofdisease is not lethal and mice do not reach a score of 5. Theaforedescribed scale may be modified to include a score of “0.5” whichis given to mice that show the earliest signs of EAE but that do notexhibit complete paralysis of the tail. Mice given a score of 0.5exhibit some or all of the following symptoms: overnight weight loss of1–2 grams; noticeable tremor when held up by the tail; and weakness atthe distal tip of the tail.

The median day of onset of EAE is determined by Kaplan-Meier Survivalanalysis. Significant differences in onset between groups are assessedusing a Log-Rank comparison. Fischer's exact test is used to analyze thestatistical significance of differences in the incidence of EAE amongthe groups of mice.

EXAMPLE 20 Mouse Cuprizone-Induced Demyelinating Disease Model

This example describes a mouse model (cuprizone-induced demyelinatingdisease or CIDD) that is designed to mimic a type of demyelination thatoccurs in some cases of multiple sclerosis referred to as primarydemyelination. CIDD is induced by feeding cuprizone(bis-cyclohexanone-oxaldihydrazone, a copper chelator) to micesubstantially as described by Matsushima et al. (Brain Pathol. 11:107,2001). At low doses of cuprizone, mature oligodendrocytes in the CNS arespecifically insulted and they become unable to provide support formyelin. Demyelination occurs when the damaged myelin is stripped fromthe axons by microglia.

Some advantages of the CIDD model are that it reproducibly results inmassive demyelination in a large area of the mouse brain and it isreversible if cuprizone is removed from the diet. The model appears wellsuited for profiling gene expression during various stages ofdemyelination and remyelination. The model has been established inC57BL/6 mice, so it is also suitable for use in KO (knockout) or Tg(transgenic) mice with the B6 background. However, there are no obviousclinical signs associated with the demyelinating process, so analysismust be done by histology.

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